bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholinergic regulation of epithelial Na+ channel (ENaC) in alveolar type 2 cells.
Authors: Takemura Yoshizumi , Helms My N. , Self Julie , Ramosevac Semra , Eaton Douglas C.
Published: June 2021

We and others have shown that epithelial Na+ channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension and by dopamine. Acetylcholine (Ach) receptors in bronchial smooth muscle are well known therapeutic targets in certain respiratory diseases, but there have been few reports on whether cholinergic agonists alter sodium transport in airway epithelial cells; therefore we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells and found that muscarinic agonists such as carbachol and oxotremorine activate ENaC. Rat AT2 cells were isolated by elastase treatment and were placed in primary culture. Cell attached‐patch clamp recording were used to investigate ENaC activities measured as the number of channels per patch times the open probability, NPo.

Results of western blotting suggested M1 and M2 receptors were present in rat AT2 cells, but nicotinic receptors were not. Carbachol and oxotremorine increased NPo of ENaC in a dose‐dependent manner but nicotine did not. Atropine almost completely blocked ENaC activation by carbachol or oxotremorine.

Our results showed that ENaC can be activated in rat AT2 cells by muscarinic agonists acting through M1 and M2 cholinergic receptors.
Extracellular chloride regulates ENaC gating
Authors: Collier Daniel M , Snyder Peter M
Published: June 2021

The extracellular domain of the epithelial sodium ion channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We tested whether chloride alters ENaC activity. In Xenopus oocytes expressing human αβγENaC, amiloride sensitive current was increased when chloride was replaced with sulfate (238% increase). Substitution of chloride with iodide or bromide resulted in an 11.6% increase and a 17.7% decrease in amiloride sensitive current, respectively. We hypothesized that chloride might regulate ENaC activity by altering channel gating. Consistent with this hypothesis, we found that changes in extracellular chloride had little effect on ENaC activity in the presence of the DEG mutation in the β subunit (βS520K) that locks the channel in the open state. We found that the response to chloride was altered by mutations in the extracellular domain that are homologous to the chloride binding site of the chicken ASIC1 crystal structure (2QTS). Mutation of αH418A and βR388A, both independently and jointly, decreased the response to extracellular chloride, while γH396A had little effect. Our results demonstrate that chloride regulates ENaC activity, providing a potential mechanism by which changes in extracellular chloride might modulate epithelial Na+ transport.
Nedd4‐2 and Nedd4 mediate degradation of TRPV6 and TRPV5
Authors: Zhang Wei , Na Tao , Peng Ji‐Bin
Published: June 2021

Nedd4 and Nedd4‐2 are archetypal HECT ubiquitin E3 ligases that dispose target proteins for degradation. Because of the proven roles of Nedd4‐2 in degradation of membrane proteins such as epithelial Na+ channel (ENaC), we examined the effect of Nedd4‐2 and Nedd4 on the epithelial Ca2+ channels TRPV6 and TRPV5, which are involved in transcellular Ca2+ transport in the intestine and kidney, respectively, using Xenopus laevis oocyte system. Co‐expression of TRPV6 with Nedd4‐2 or Nedd4 resulted in a 47.9±1.4% or 28.3±1.8% reduction in TRPV6‐mediated Ca2+ uptake. Similarly, a 51.9±1.7% or 26.2±4.8% decrease in TRPV5‐mediated Ca2+ uptake was observed when Nedd4‐2 or Nedd4 was co‐expressed. Corresponding decrease in TRPV6‐ or TRPV5‐mediated Na+ current by Nedd4‐2 was detected using voltage clamp technique. Western blot analysis revealed that TRPV6 and TRPV5 protein were decreased by Nedd4‐2 and Nedd4, respectively, in a dose‐dependent manner. The down‐regulation of TRPV6 by Nedd4‐2 appeared to be dependent on the ubiquitin ligase activity, because the C922S ubiquitin ligase deficient mutant of Nedd4‐2 failed to inhibit TRPV6. Furthermore, an increase in TRPV6‐mediated Ca2+ uptake was observed when the HECT domain of Nedd4‐2 was removed. The Ca2+ binding C2 domain of Nedd4‐2 was also dispensable in this regulation, because the Nedd4‐2 construct without C2 domain exhibited a stronger inhibitory effect on TRPV6 than the wild‐type Nedd4‐2. In conclusion, our data suggest that Nedd4‐2 and Nedd4 may be involved in the degradation of TRPV6 and TRPV5.
Identification of Phosphorylation‐Dependent Binding Partners of Aquaporin‐2 (AQP2) By Protein Mass Spectrometry
Authors: Zwang Nicholas , Hoffert J , Pisitkun T , Moeller H , Fenton R , Knepper M
Published: June 2021

Phosphorylation of Ser‐256 at AQP2's cytoplasmic COOH‐terminus is well‐accepted as a key step for regulated AQP2 trafficking. Here we identify binding partners to phosphorylated and nonphosphorylated forms of the AQP2 COOH‐terminus using mass spectrometry. Cytosol from inner medullary collecting ducts (IMCDs) isolated from rat kidneys was incubated with biotinylated "bait" peptides, representing the COOH‐terminal AQP2 tail in its nonphosphorylated and phosphorylated forms, to capture differentially‐bound proteins prior to LC‐MS/MS analysis and immunoblotting. We found and confirmed 7 proteins that bound preferentially to the nonphospho‐peptide (hsc70, hsp70‐1 and ‐2, annexin II, PP1c, GDI‐2, and RAN) and one protein that bound preferentially to the S256 phosphopeptide (hsp70‐5 or BiP). AQP2 interactions with hsc70, hsp70‐1 and ‐2, as well as annexin II were previously identified. Immunoprecipitation of AQP2 from native IMCD lysates confirmed interactions with annexin II, PP1c, and BiP. Immunogold EM studies demonstrated that BiP is present not only in the ER, but in the cytoplasm and apical plasma membrane of rat collecting duct cells. Furthermore, confocal immunofluorescence showed partial colocalization of BiP with AQP2 in non‐ER compartments. These results suggest that phosphorylation of AQP2 at Ser‐256 may regulate binding of hsp70 family proteins, including BiP, to the COOH‐terminal tail.
2‐D DIGE ‐LC/MS/MS analysis of proteins which interact with heat shock factor 1 in liver during sepsis
Authors: Jao Hsiao‐Ching , Lai Yun‐Wei
Published: June 2021

Heat shock factor 1 (HSF1) is the major transcriptional factor for heat shock proteins. In sepsis animal model, there was much higher mortality rate in HSF1 knock out animals. HSF1 does play an important survival role in sepsis. Via experimental sepsis model by cecum ligation and puncture (CLP), there was increased HSF1 expression in nuclear fraction of hepatocytes during early sepsis, and decreased during late sepsis. In respecto of protein‐protein interaction with HSF1, nuclear proteins were immunoprecipitated with anti‐HSF1 antibody and analyzed by 2‐D difference gel electrophoresis (DIGE). There were 23 proteins dots revealed by Decyder BVA with significant changes in sepsis. 7 protein dots of them have been identified by LC/MS/MS, including protein dots 267 (grp75), protein dots363 (HSP60), protein dots 417 (ATPase F1 alpha subunit), protein dots 435 (Lipoamide acyltransferase component of branched‐chain alpha‐deto acid dehydrogenase complex), protein dots 572 (Purine‐rich single‐stranded DNA‐binding protein alpha isoform 1), protein dots 621 (2‐oxoisovalerate dehydrogenase beta subunit) and protein dots 651 (peroxisomal trans‐2‐enoyl‐CoA reductase). These proteins interacted with HSF1 may participate in heat shock protective mechanism during sepsis.
Glycogenin protein levels in sub‐cellular fractions of different muscle fiber types from stimulated rodent hind limb following changes in glycogen content
Authors: Wilson Rhonda Joanne , Yuan Zongfei , Graham Terry E
Published: June 2021

Glycogenin (GN) is bound to glycogen. At rest, GN distribution mimics glycogen (cytosolic/ membrane‐associated). GN may also co‐localize with actin. GN distribution across muscle fiber types following contraction was examined. One hind limb (40 F Sprague Dawley rats) was stimulated for 40 min to lower glycogen. Unstimulated and stimulated soleus (SOL), red (RG), and white (WG) gastrocnemius muscles were separated into sub‐cellular fractions [pellet 1/supernatant 1= P1/S1; S1 separated into S2 (cytosolic) + S3 (membrane/glycogen) + P3 (cytoskeletal)] and examined for total and unglycosylated GN levels. Tissue (corresponding unstimulated and stimulated) was grouped (n=6) by post‐stimulation glycogen level (WGI <40; WGII <100; RGI <100, RGII >100; SOL >100 μmol▸ dry wt.‐1). At rest, ~50% of GN was located in each P1 and S1. In S1, ~4, 87, and 9% of GN was located in S2, S3 and P3 fractions, respectively. Post‐stimulation, GN increased in S2 and decreased in S3 and P3 fractions (P≤0.05). <25% of GN in S2 was unglycosylated, suggesting re‐distribution of mostly small granules. In WGI, however, glycogen content decreased the most and the distribution of total GN (and glycogen synthase) in the P1 fraction increased. Low glycogen content in WG may lead to sequestering of GN (and GS) in the P1 fraction (potentially with actin) for glycogen re‐synthesis following acute stimulation in rodents. Funded by NSERC of Canada.
Exercise training maintains normal HPA feedback and prevents hypercortisolemia in the ZDF rat
Authors: D'souza Anna Marie , Campbell Jonathan , Vranic Mladen , Kiraly Michael , Riddell Michael
Published: June 2021

Zucker Diabetic Fatty (ZDF) rats develop type 2 diabetes (T2D), resulting in elevations in circulating corticosterone (CORT) concomitantly with the onset of hyperglycaemia. We hypothesize that prolonged training results in increased negative feedback sensitivity of the hypothalamic‐pituitary‐adrenal axis, thus maintaining normal CORT values. At 5 wks of age, rats were assigned to either: basal (B), exercise (E), or sedentary (S) groups. E was given free access to running wheels for 10 wks. Plasma CORT and intraperitoneal glucose tolerance test values were collected prior to sacrifice at age 6 wks in B, and 16 wks in S and E. Food consumption and weight gain were similar among S and E, despite E running 4‐7 km/day. S demonstrated a T2D phenotype by 12 wks of age (fasting glycemia >11 mM), while E had fasting glucose levels comparable to B (both groups <6 mM). S also expressed higher plasma CORT values compared to E (129.9±43.7 vs. 33.3±7.3 ng/ml, p<0.05) or B (25.6±5.9, p<0.05). S had lower hippocampal glucocorticoid receptor (GR) expression than E (0.64±0.11 vs. 1.11±0.06, p<0.05) and B (1.00±0.09, p<0.05). GR expression in the pituitary gland was not different between groups. This data suggests that exercise training may prevent the hypercorticosteronemia and hyperglycemia associated with sedentary behaviour, likely through maintained protein expression of hippocampal GR. This study was funded by CIHR and NSERC.
Heat shock protein 72 stabilizes the mitochondrial membrane potential in the anoxia tolerant turtle (Trachemys scripta) neuron
Authors: Milton Sarah Louise , Kesaraju Shailaja
Published: June 2021

This study was undertaken to determine if Heat Shock Protein 70 (Hsp72), which doubles in the anoxia tolerant freshwater turtle brain, provides protection against cell death. The turtle survives both extended anoxia and reoxygenation, through such protective responses as alterations in heat shock proteins and increases in adenosine and GABA, and by suppression of reactive oxygen species (ROS). Neuronal cell cultures were transfected with specific siRNA against Hsp72 and apoptosis investigated by Western blot. ROS production was measured by Amplex Red, and the mitochondrial membrane potential (MMP) by JC‐1. Hsp72 in vitro increased 2X over basal by 16h. SiRNA treatment knocked down Hsp72 by 60%. Cell death doubled in anoxic and A/R cells; the Bcl‐2/Bax ratio decreased from 1.2 in controls to 0.8 in knockdown cultures. Cyt C and caspase‐3 levels did not change, although AIF increased. H2O2 release though increased 6‐7‐fold upon reoxygenation compared to controls, and preliminary evidence suggests an organelle‐associated localization for Hsp72 which could be mitochondrial. The MMP does not recover in knockdown cells upon reoxygenation following anoxic depolarization, suggesting that in the anoxia tolerant neuron, Hsp72 does not prevent cell death by directly inhibiting the intrinsic apoptotic pathway but instead stabilizes the mitochondrial membrane itself.

Research supported by an NIH R‐15 grant to SLM.
N‐acetylcysteine is an effective ex vivo treatment of oxidative stress in SEPN1 – related myopathy
Authors: Arbogast Sandrine , Ferreiro Ana
Published: June 2021

Selenoprotein N (SelN) defects are responsible for a congenital muscle disorder (SEPN1‐related myopathy) for which no treatment is available. We have recently established that human cells devoid of SelN show abnormal susceptibility to oxidative stress, increased intracellular oxidant activity and excessive protein oxidation. Therefore, SelN plays a role in cell protection against oxidative stress. We hypothesized that oxidative stress in SelN deficiency can be effectively targeted by antioxidants, and developed an ex vivo model to test potential‐therapeutic drugs using muscle cells and fibroblasts from patients carrying SEPN1 null mutations. We analyzed the susceptibility of SelN‐devoid fibroblasts to H2O2 before and after pre‐treatment with the antioxidants astaxanthin, fisetin and N‐acetyl cysteine (NAC). Astaxanthin and fisetin had a partial protective effect. In contrast, NAC protected SelN‐devoid fibroblasts and muscle cells from cell death in OxS conditions and normalized the levels of oxidized proteins in SEPN1‐mutant myotubes. NAC efficiency on mutant cells suggests that this thiol‐donor targets more directly the pathway altered by SelN deficiency. This study establishes for the first time that antioxidants can effectively target the primary defect in this genetic disorder, thus representing the first specific pharmacological treatment for a congenital myopathy. Supported by AFM.
Cardiac autonomic function in mice expressing dominant‐negative mutation of PPAR‐gamma (PPARG) in vascular smooth muscle
Authors: Borges Giulianna da Rocha , Sigmund Curt D
Published: June 2021

Mice expressing dominant‐negative PPARG in vascular smooth muscle (S‐P467L) exhibited vascular dysfunction, hypertension and tachycardia. We hypothesize that a high sympathetic outflow contributes to cardiovascular dysfunction observed in these mice. S‐P467L and non‐transgenic (NT) littermates were instrumented with radiotelemeters. Basal arterial pressure and heart rate (HR) were continuously recorded. Spontaneous baroreflex (BR) sensitivity was determined using the sequence method. S‐P467L mice exhibited hypertension (116±10 vs 101±3 mmHg) and tachycardia (578±10 vs 487±10 bpm) compared with NT. S‐P467L mice showed an elevated cardiac sympathetic tone, as demonstrated by increased bradycardic response to propranolol (‐102±20 vs ‐43±8 bpm) and a decreased cardiac vagal tone as characterized by decreased tachycardic response to methyl‐atropine (44±12 vs 78±8 bpm). The intrinsic HR, calculated after blockade of vagal and sympathetic outflow to the heart, was elevated in S‐P467L mice compared to NT (511±11 vs 476±11 bpm). BR function was also impaired in S‐P467L mice as shown by the decreased gain (1.82±0.26 vs 2.95±0.34 ms/mmHg) and BR effectiveness index (5±1 vs 8.2±0.7%). These results suggest that the cardiac dysfunction caused by the interference with PPARG in vascular smooth muscle could be caused by a combination of factors such as autonomic imbalance, impaired BR and increased intrinsic HR.
Medical and premedical scientific competencies: opportunity and challenge
Authors: Silverthorn Dee U , Galey William R. , Dalley Arthur E. , Bond Judith S
Published: June 2021

In 2007 the AAMC and Howard Hughes Medical Institute convened the Scientific Foundations for Future Physicians Committee (SFFP). The committee, composed of basic and clinical scientists and undergraduate scientist‐educators, was charged with "recommend[ing] specific [scientific] competencies . . . fundamental to medicine that all medical students should demonstrate," and "identify[ing] scientific competencies that learners should demonstrate before entry into medical school [with] emphasis . . . on defined knowledge, scientific concepts and skills rather than specific courses or disciplines." The committee report, published this spring, begins with overarching principles that span all of the scientific disciplines, followed by scientific competencies and associated learning objectives for medical and premedical students. The premedical section ends with a discussion of how innovative curricula might allow students to achieve these competencies in place of the traditional pre‐med coursework. The SFFP report debuts as AAMC begins revision of the MCAT, the National Board of Medical Examiners revises the USMLE, NSF discusses the undergraduate biology experience in its Vision and Change meetings, and the College Board revises the AP Biology curriculum. Coordinating recommendations from these diverse groups will provide faculty with opportunities and challenges in the years to come.
Preliminary studies of myosin isoforms in the masseter muscle of mice fed a soft diet
Authors: Druzinsky Robert E. , Adamczyk Agnieszka , Hennessy Sara
Published: June 2021

Plasticity in myosin isoform expression resulting from increases and decreases in functional muscle activities is well known. Expression of myosin isoforms changes according to a rigid format (Schiaffino and Reggiani, 1996; Reader et al., 2001):

I/alpha <‐‐‐‐> IIa <‐‐‐‐> IIx <‐‐‐‐> IIb. Adult mice were fed a soft diet (standard mouse diet in powdered form) for ten days as a model of disuse atrophy of the jaw closing muscles. Masseter muscles were dissected free, blotted dry, and snap frozen in isopentane cooled in liquid nitrogen. Ten micron sections were cut on a cryostat and stained using monoclonal antibodies to myosin heavy chain isoforms (BA‐G5, 10F5, 6H1, 2F7, SC‐71, A4.840). No fibers positive for A4.840 (Type I) were found in either control or experimental masseters. Although the masseter is composed entirely of type II fibers, some fibers are labeled by BA‐G5 (alpha). Preliminary results suggest that BA‐G5 expression is increased in the masseter muscles of animals fed the soft diet. Thus, the masseter shifts toward the type I phenotype without actually producing a true type I isoform. This result is in accord with Kitagawa et al. (2004) who found increased numbers of type IIa fibers in the masseter of rabbits fed a soft diet.

Supported by a Governors State University Research Grant and NIH P20 MD001816.
Cellular and subcellular localization of N‐terminally phosphorylated NCC in normal rat kidney
Authors: Pedersen Nis Borbye , Nielsen Søren , Fenton Robert A
Published: June 2021

N‐terminal phosphorylation of the renal thiazide‐sensitive NaCl cotransporter NCC (or TSC) (Slc12a3) is proposed to play a major role in its transport activity and cellular localization, as seen for its Slc12‐family homologues NKCC1 and NKCC2. We generated novel phospho‐specific antibodies against two N‐terminally conserved phosphorylation sites (Thr53, Thr58 and Thr53/Thr58 together) of NCC. Dot blot analysis confirmed the phospho‐specificity of the antibodies. Immunohistochemistry showed distinct staining of the apical plasma membrane domain of distal convoluted tubule (DCT). Confocal microscopy with specific tubule markers confirmed phosphorylated NCC (pNCC) was localized predominantly to DCT. At the transition between DCT and connecting tubule, pNCC was also detected, but in non‐AQP2 containing cells. Using immunogold electron microscopy, pNCC (Thr53 and Thr58) was localized only to apical plasma membrane domains and was not observed within the cell. Immunoblot analysis of rat kidney homogenates showed a protein band of 160 kDa in cortex, the expected size for glycosylated NCC. Acute vasopressin exposure increased pNCC abundance approximately 300%. Studies in Brattleboro rats confirmed that vasopressin regulates NCC phosphorylation in the DCT.
Oxidant status is maintained through different mechanisms in male and female intrauterine growth restricted offspring
Authors: Hennington Bettye Sue , Dawson Antionette , Ojeda Norma , Royals Thomas B. , Alexander Barbara T.
Published: June 2021

Placental insufficiency in the rat leads to low birth weight with development of hypertension in male intrauterine growth restriction (IUGR) offspring; however, female IUGR are normotensive indicating sex differences in response to fetal insult.We previously reported that renal superoxide production is significantly elevated in male IUGR versus male Control, an increase abolished by treatment with TEMPOL (1mMol/L for 2 weeks).Furthermore, chronic tempol also abolishes hypertension in male IUGR suggesting oxidative stress contributes to hypertension in male IUGR.In this study we tested the hypothesis that oxidant status is maintained through different mechanisms in male and female IUGR; thus, leading to sex differences in IUGR blood pressure.We examined antioxidant enzymes CuZn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and the mitochondrial enzyme, manganese SOD (MnSOD).We observed protein expression of CAT, GPXe, and MnSOD were significantly decreased in hypertensive male IUGR as compared to normotensive female IUGR, a decrease of 40%, 53%, and 51%, respectively. These studies indicate that sex differences in IUGR blood pressure may involve an imbalance of anti‐oxidant enzymes.NIH/NCRR INBRE RR016476, NIH/NCMHHD IP20MD002725‐01, NIH RO1‐HL074927‐01A1, NIH/NHLBI PO1‐H51971, NIH/NHLBI RO1‐HL38499.
The N‐terminal region of BRG1 contains protein interaction domains that are required for SWI/SNF activity
Authors: Trotter Kevin W. , Archer Trevor K.
Published: June 2021

The packaging of eukaryotic DNA into chromatin creates an impediment to the process of transcription by hindering binding of essential factors required for transcriptional initiation. BRG1, the central ATPase of the human SWI/SNF‐BAF complex, has been shown to be a critical factor for the functional activity of nuclear receptor complexes. Studies using BRG1 truncation and deletion mutants suggest the chromatin remodeling protein contains functional motifs important for transcriptional regulation. To further examine these conserved domains we performed a yeast‐two hybrid study using baits spanning the BRG1 N‐terminus to identify proteins that may be required for SWI/SNF activity. Several proteins were found to associate within the N‐terminal region of BRG1 including BRD7, a regulator of cell cycle progression, CBX5, which is involved in heterochromatin maintenance, and transcriptional regulators ARA160 and SSXT, as well as proteins with identified roles in apoptosis, replication and DNA repair. To verify these potential BRG1 interacting proteins we performed co‐immunoprecipitation studies using various human cell lines. Subsequently, we will perform siRNA knockdown assays to determine if these BRG1 associating proteins are required for SWI/SNF activity including nuclear receptor‐mediated transcriptional activation from endogenous chromatin loci.
TP508, a thrombin‐derived peptide, modulates TLR‐4 dependent cytokine expression in differentiated THP‐1 cells
Authors: Rousseau Emma , Smoke Chris , Zepp Jarod , Dinarello Charles A. , Sheller Michael R.
Published: June 2021

Accelerated wound healing by TP508, a thrombin‐derived synthetic peptide, may involve inflammatory mediators. The mechanism of action for TP508 is hypothesized to be integrin binding at its RGD site. THP‐1 monocytes were differentiated to an adherent macrophage phenotype with 50 ng/mL phorbol 12‐myristate 13‐acetate (PMA). Cells were pretreated with TP508 for 30 minutes, followed by lipopolysaccharide (LPS) for 3.5 hours. TNFα, IL‐8 and IL‐1 β were analyzed via ELISA. TP508 decreased expression of TNFα and IL‐8 and increased IL‐1 β expression independently of LPS. When heparin‐sulfate was used to stimulate TLR4 receptors, TP508 again reduced TNFα expression.

Given the peptide sequence of TP508, direct blocking of the TLR4 receptor is improbable, so the TP508 RGD site was explored for potential interaction with TLR4‐dependent cytokine stimulation. Variants of TP508 were used to test whether the RGD‐integrin binding was responsible for the reduction in cytokine expression in these cells. An αV β3 integrin blocking antibody was as effective in reducing LPS‐stimulated cytokine expression as the TLR4 activity‐blocking antibody. Treatment of cells with αV β3 integrin‐binding peptide GRGDSP also significantly reduced TNFα expression in response to LPS. This preliminary evidence suggests that TP508 may modulate cytokine expression through an integrin dependent mechanism.
The interplay between microRNA‐21 and endothelin receptor B (EDNRB) in gastric cancer
Authors: David Stefan , Olaru Alexandru V , Paun Bogdan C , Jin Zhe , Meltzer Stephen J. , Selaru Florin M
Published: June 2021

Background

MicroRNAs (miRs) are short noncoding RNA species that bind to mRNAs reducing the expression of corresponding proteins. Endothelins (ET 1‐3) are involved in vasoconstriction, angiogenesis, mitogenesis and apoptosis acting through the endothelin receptors EDNRA and EDNRB.

Methods

7 normal gastric (N) and 3 gastric cancer (GC) tissues were used for microRNA arrays. Global miR profiles were analyzed by Significance Analysis of Microarrays. MiR results were confirmed by qRT‐PCR on additional 72 gastric tissues. TargetScan identified EDNRB as a putative miR‐21 target. The EDNRB expression was evaluated by qRT‐PCR on all 82 gastric tissues. MKN28 GC cells were transfected with miR‐21 inhibitor (mir21i) and EDNRB level was assessed by western blotting.

Results

The array data showed a 6‐fold miR‐21 upregulation in GC vs. N. qRT‐PCR confirmed the miR‐21 upregulation GC (p=0.011). qRT‐PCR also showed that EDNRB expression was 1.9 times lower in GC (p=0.015) vs. N, suggesting that EDNRB may function as a tumor suppressor, potentially downregulated by miR‐21. Confirming this hypothesis, miR‐21 inhibitor (miR21i) enhanced EDNRB protein in the MKN28 cells (Fig.1).

Conclusion

our data confirm miR‐21 overexpression in a large cohort of GC and indicate, for the first time, that EDNRB is a miR‐21 target in GC. Our findings suggest that miR‐21 plays an etiologic role in GC partly through EDNRB regulation. Lastly, EDNRB may deserve further study as a tumor suppressor in GC.
Apigenin protects HT22 murine hippocampal neuronal cells against ER stress‐induced apoptosis
Authors: Choi A Young , Kang Insug
Published: June 2021

Endoplasmic reticulum (ER)‐stress is known to induce neuronal cell death and is involved in neurodegenerative diseases including Alzheimer's diseases, Parkinson diseases, and cerebral ischemia. Apigenin, a bioactive flavonoid, is shown to have anti‐tumorigenic and anti‐inflammatory properties in various cell types. In the present study, we investigated the effect of apigenin on ER stress‐induced apoptosis and action mechanism involved in murine HT22 hippocampal neuronal cells. Apigenin blocked apoptotic cell death of HT22 cells induced by ER stress inducers, thapsigargin (TG) and brefeldin A (BFA). We showed that apigenin reduced the expression levels of unfolded protein response (UPR) proteins including C/EBP homologous protein (CHOP) and glucose response protein 78 (GRP78) and phosphorylations of MAP kinases in TG or BFA‐treated HT22 cells. We also showed that TG or BFA increased intracellular ROS accumulation and apigenin blocked ROS accumulation induced by TG or BFA. Moreover, we found that TG or BFA decreased the mitochondrial membrane potential (MMP) and apigenin inhibited this decrease in MMP by TG or BFA. These results suggest that apigenin inhibits ER stress‐induced apoptosis by reducing ROS accumulation, mitochondrial dysfunction, and CHOP induction in HT22 hippocampal neuronal cells.

This work was supported by grants from KOSEF of Korean Government.
Expression and Purification of LpPLA2 for Crystallography
Authors: Matico Rosalie Elizabeth , Myers Linda , Taylor David , Sweitzer Sharon , Elkins Patricia , Smallwood Angela , Pietrak Beth , Schwartz Benjamin , McCormick Patricia , Johanson Kyung
Published: June 2021

Lipoprotein‐associated lipase (LpPLA2) is a 45kD calcium independent lipase secreted by macrophages, T cells, and mast cells in atherosclerotic plaques. LpPLA2 is mainly associated with ApoB on LDL particles and is responsible for hydrolyzing the sn‐2 chain of oxidatively modified phospholipids resulting in lysophatidylcholine and non‐esterified fatty acids ‐ both inflammatory mediators. Crystallography of LpPLA2 would be beneficial for inhibitor optimization. LpPLA2 was expressed by baculovirus with a dual N‐terminal his/Streptavidin binding protein (SBP) tag and a TEV cleavage site engineered prior to residues 47‐429. No signal sequence was engineered into the construct; however, active LpPLA2 was detected at high levels in both the medium (~35mg/L) and cells (~15mg/L). His/SBP/TEV(GS)LpPLA2 (47‐429) was captured from the medium by Ni Sepharose, the his/SBP/TEV(GS)LpPLA2(47‐429) in the imidazole eluate was captured onto Streptavidin resin and (GS)LpPLA2 (47‐429) was released by on column TEV cleavage. (GS)LpPLA2(47‐429) was further purified by Blue‐Sepharose. Mono Q was used for high resolution chromatography and detergent exchange. Final product demonstrated full active site accessibility by titration with GSK184919A which acylates the active site serine with a dimethylamido group resulting in a +71Da mass shift. A surface cysteine was susceptible to cysteinylation and this appeared to have no impact on activity. Crystals of the final product formed within a day and the apo‐structure was solved to ~2.0? resolution.
Long‐time influence of copper ions on the wine yeast populations increases number of resistant morphs
Authors: Shatirishvili Aivengo , Machavariani Nino , Zarnadze Tea
Published: June 2021

Natural populations of the wine yeast (Sacch. cerecisiae var. vini) have been exposed to copper‐containing pesticides for a long time. The strains belonging to two different populations of the wine yeast were investigated on their resistance. One of the populations was 'Rqatsiteli', that had continuously been exposed to copper ions for many years, but the other ('Izabela') had never been treated with the pesticides. The strains were isolated from the wine sediment. 250 strains from each population were analyzed and 10 of them belonged to micropopulations. The 'Rqatsiteli' strains were found to be heterogeneous: when growing on the medium supplemented with 1‐7mM copper ions, they quitted growing in asynchronous manner. Existence of three morphs was stated: 'sensitive' ‐ 31.6%, 'mild' ‐ 38.8% and 'resistant" ‐ 29.6%. 'Izabela' population expressed less resistance when compared to "Rqatsiteli' strains and varied within the range of 1‐5 mM with only two morphs among them: 'sensitive' ‐ 63.6% and mild ‐ 36.4%. Genetic analysis in 10 strains for each population was performed. The strains of 'resistant' morphs contained two active resistance‐determining loci while the others ‐ just one. The both populations were polymorphic if evaluated by the trait under study. Driven by natural selection, number of the strains resistant to copper ions were increased.
Cerebrospinal fluid proteomics in tuberculous meningitis patients
Authors: KATARIA JITENDER , Rukmangadachar Lokesh Ankenahally , Hariprasad Gururao , Kaur Punit , Tripathi Manjari , Singh Tej P , Srinivasan Alagairi
Published: June 2021

Background

Tuberculous meningitis is a grave extra‐pulmonary complication of tuberculosis caused by Mycobacterium tuberculosis group of bacteria. Tuberculous meningitis comprise of ~5% of all cases of tuberculosis worldwide. The diagnosis is conventionally made by Acid fast bacilli (AFB) smear, culture and polymerase chain reaction (PCR) of cerebrospinal fluid for tb bacilli gene sequence. We used proteomic technologies to discover protein biomarkers for the disease.

Methods

We compared the cerebrospinal fluid of patients of tuberculous meningitis with normal healthy controls using proteomic technologies. All the patients were HIV negative and culture positive, while controls had no neurological deficits and no active tuberculosis. Cerebrospinal fluid samples were separated using 2D DIGE. Biological variations of the protein expression levels were analysed with DeCyder 6.5 software and evaluated for reproducibility and statistical significance. Differentially expressed proteins were identified by tandem mass spectroscopy.

Results

The profile of differentially expressed protein revealed that between patients and controls serum albumin and its precursors, arachidonate 5‐lipoxygenase and immunoglobulin kappa chains are up regulated and apolipoprotein E and its precursor are down regulated in disease state.

Conclusions

Identification of differentially expressed proteins gives us the opportunity to understand the pathogenesis of tuberculous meningitis and these proteins can be potential biomarkers for non invasive diagnosis and prognosis of disease under treatment.

The work has been supported from grant from Department of Biotechnology, Government of India to AS.
Characterization of Human Cervical Mucous Proteome
Authors: Panicker Gitika , Ye Yiming , Wang Dongxia , Unger Elizabeth R
Published: June 2021

Cervical cancer is among the most common cancers in women worldwide. Discovery of biomarkers for early detection of cervical cancer would improve current screening practices and reduce the burden of disease. In this study, we report characterization of the human cervical mucous proteome as the first step towards protein biomarker discovery. Proteins extracted from pooled mucous were characterized using 1‐DE and 2‐DE followed by LC/MS. Post‐translational modifications like phosphorylation and glycosylation of proteins were analyzed using fluorescent stains. A total of 107 unique proteins were identified, with plasma proteins being most abundant. These proteins represented major functional categories of metabolism, immune response, and cellular transport. Removal of high molecular weight abundant proteins by immunoaffinity purification did not significantly increase the number of protein spots resolved. Each method identified some unique proteins, indicating the advantage of utilizing multiple techniques in sample preparation, fractionation, and analysis. Thus, profiling cervical mucous proteins can further our understanding of the proteome and its potential as biomarkers.
Identification of functionally important regions of adrenal and duodenal cytochromes b561
Authors: da Silva Giordano F.Z. , Liu Wen , Palmer Graham , Kulmacz Richard J
Published: June 2021

Adrenal and duodenal cytochromes b561 (cyt b561 and Dcytb, respectively) are transmembrane proteins sharing four conserved histidines that provide axial ligands to the low potential (bL) and high potential (bH) hemes; both proteins shuttle electrons from ascorbate. Spectrophotometric analyses of recombinant cyt b561 with Ala‐scanning mutations in extra‐membrane segment 3 (EM3, residues 70‐85) and cyt b561 chimeras carrying EM3, EM5, or EM7 from Dcytb were used to indentify structural features affecting the interaction with ascorbate. For cyt b561 the midpoint ascorbate concentrations (in μM) were 5.2 ± 0.4 (bH) and 318 ± 50 (bL). Significant changes in the bH midpoint were found for R74A (11.2 ±1.3), R77A (13.8 ± 1.8), and R82A (2.1 ± 0.8); the bL midpoint was altered in R74A (1022 ± 130), R77A (705 ± 179), and T83A (182 ± 39). The cyt b561 chimera with EM5 from Dcytb showed large changes in both ascorbate midpoints (28 ± 3; 873 ± 50). These results identify several functionally relevant residues in EM3 of cyt b561, and suggest that structural differences between cyt b561 and Dcytb in EM5 lead to functional differences between the two cytochromes. Support: NIH grant GM 080575.
Characterization of genotype ‐ phenotype correlations in Adenylosuccinate Lyase (ADSL) Deficiency
Authors: Zikanova Marie , Skopova Vaclava , Hulkova Helena , Kmoch Stanislav
Published: June 2021

ADSL deficiency is inherited disorder of purine metabolism affecting mainly central nervous system. Three forms differing in severity of symptoms and correlating negatively with ratios of accumulating SAdo/SAICAr in body fluids were established. The mechanisms leading to selective neuronopathy and underlying the clinical and biochemical heterogeneity are unknown.

We analysed 22 patients, identified 16 ADSL mutations, expressed and characterized catalytic properties of recombinant wild type and mutant proteins, investigated expression patterns of ADSL in available tissues from 3 patients and correlated these data with patients' clinical and biochemical presentation.

We found that phenotype severity corresponds with residual enzyme activity (calculated as a mean of homoallelic activities), but the mutant enzymes displayed proportional decrease in activity to both of its substrates, so no ground for the varied SAdo/SAICAr ratios was found.

Western blot and immunohistochemistry analyses showed in patients generally reduced amounts of ADSL in investigated tissues.

Our results suggest that selective affection of central nervous system is caused by toxic effects of accumulating SAdo‐SAICAr. Phenotype's heterogeneity may be explained by differential activity of metabolite transport across blood‐brain barrier in individual cases.

This work was supported by grants GACR 301070600 and MSM CR 20620806.
Identification of an ENU mutant mouse with liver fibrosis
Authors: Chen Yi‐Ting , Chang Mei‐Ling , Kung John T.
Published: June 2021

With the human genome sequenced, the race is on to understand the functions of the 30,000 or so genes. Ethyl‐nitrosourea (ENU) was used to treat C57BL/6 (B6) male mice, followed by a 3‐generation breeding scheme to generate G3 mice that were screened for immunological phenotypes under recessive genetic control. The primary phenotype of one mutant, P321, was elevated serum AST/ALT. Major organs of affected mice were subjected to pathology analysis and striking liver‐specific fibrosis was found. Recessive mode of inheritance was confirmed by mating an affected G3 male to wildtype B6 females to generate F2 offspring. No affected phenotype was found in F1 mice and approximately 1 in 4 F2 mice were pheno‐deviants, consistent with monogenic control. Affected G3 mice were outcrossed to generated F2 offspring and the affected F2 mice were subjected to linkage analysis by haplotype interval mapping (Neuhaus and Beier, 1998. Mammalian Genome 9, 150‐154). The mutant gene was found to be linked to chromosome 5. The P‐321 mutant may be used as a model to study the molecular mechanism of liver fibrosis and to establish ways to treat, prevent, or delay the onset of such diseases.
How Neurons Internalize Alzheimer's Disease Amyloid β Protein
Authors: Worthy Patrice N , Olenych Scott G , Fulzele Smita , Davidson Michael W , Poduslo Joseph F , Kandimalla Karunya K
Published: June 2021

Objective

The objective is to investigate cellular mechanisms mediating the internalization of beta‐amyloid 40 (Aβ40) in part of the neurovascular unit, neurons, and blood‐brain barrier (BBB) endothelial cells.

Methods

Localization of fluorescein labeled Aβ40 (F‐Aβ40) and other endocytotic markers in wild type (WT) mouse brain slices, PC12 cells, rat primary hippocampal (RPH) neurons, and bovine brain microvascular endothelial (BBME) cells was investigated using laser confocal or fluorescence microscopy. The uptake of fluorophores in these cells was quantified by flow cytometry.

Results

Laser confocal micrographs of WT mouse brain slices treated with F‐Aβ40 demonstrated selective accumulation of the protein in cortical and hippocampal neurons via non‐saturable, energy independent, and non‐endocytotic pathways. A significant portion of internalized Aβ40 is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of Aβ40, and accumulate the protein in acidic cell organelles, indicative of endocytotic uptake.

Conclusion

The difference in internalization of Aβ40 between neurons and BBB endothelial cells may provide clues to understanding how various cells can regulate Alzheimer's disease (AD) proteins and help explain the vulnerability of cortical and hippocampal neurons to AD toxicity.
The recycling ceramide is a precursor of hypertonicity induced increase of glucosylceramide synthesis
Authors: Pescio Lucila Gisele , Sterin‐Speziale Norma Beatriz
Published: June 2021

We have reported that hypertonicity induces MDCK cell differentiation by a GlucosylCeramide Synthase (GCS) dependent mechanism. Ceramide substrate can stem from the de novo synthesis or the recycling pathway. In this study we evaluate the involvement of both pathways in the hypertonicity induced increase of Glycosphingolipid (GSL) synthesis. Confluent MDCK cells were submitted to high NaCl concentration or kept under isotonicity. GSL metabolism was determined by using 14C‐Galactose (14C‐Gal) and 14C‐Palmitic Acid (14C‐PA) as radioactive precursors in the presence of Fumonisin B1 (FB1), a ceramide synthase inhibitor, or Cycloserine (CS), a serine palmitoiltransferase inhibitor. Under isotonic conditions, 14C‐Gal/GlcCer formation was FB1/CS sensitive, while hipertonicity induced a 14C‐Gal/GlcCer increase partially sensitive to FB1 but not to CS. In control cultures, 14C‐Gal/GlcCer accounted for 23% of total GSLs formed, while 14C‐PA/GlcCer represented 44% of total GSLs. Over 24h of hypertonicity, 14C‐Gal/GlcCer synthesis increased to 43%, while 14C‐PA/GlcCer levels decreased significantly. The results suggest that the pool of Cer involved in the hypertonicity induced MDCK cell differentiation comes from the recycling but not from de novo synthesis pathway. This work was supported by the University of Buenos Aires (UBACyT B068), CONICET (PIP 5557) and ANPCyT (PICT 33154) grants.
Insulin‐mediated redox regulation of cyclic AMP‐dependent protein kinase (PKA) in rat adipocytes
Authors: Riveros‐Rosas Héctor , Zentella de Piña Martha , Vázquez‐Meza Héctor , Pardo Juan Pablo , Rendón Juan Luis , Villalobos‐Molina Rafael , Piña Enrique
Published: June 2021

Lipolysis and lipogenesis are two highly regulated pathways. Catecholamines promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. H2O2 mimics insulin action inhibiting lipolysis, and insulin stimulates NADPH oxidase in adipocytes and transiently generates H2O2. Therefore, the possibility that H2O2 might regulate cAMP‐dependent protein kinase (PKA) was explored.

Results show that H2O2 generated by insulin in rat adipocytes impaired cAMP‐mediated amplification cascade of lipolysis. Micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of both type IIß or type IIα cyclic AMP‐dependent protein kinase (PKA) holoenzyme. However, H2O2 was ineffective on reconstituted PKA holoenzyme preincubated with cAMP, or if added to the catalytic αsubunit alone. The same results were obtained if the catalytic αsubunit was substituted by its C199A mutant. H2O2 inhibition of PKA activation remained after H2O2 elimination but was reverted with thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing PKA activation. Phylogenetic analysis showed that Cys‐97 is conserved only in type II (but not type I) regulatory subunits. Supported by DGAPA‐UNAM grant IN208308
Generation of a heterodimer of NGF muteins to study the function of the putative TrkA‐p75 heteroreceptor complex
Authors: Mehta Hrishikesh M , Neet Kenneth E
Published: June 2021

Nerve growth factor (NGF), a non‐covalently bound dimer, binds two distinct receptors; TrkA (tyrosine kinase receptor family) and p75 (TNF receptor family). NGF supports survival and differentiation through these receptors and can also induce apoptosis through p75 alone. NGF also exhibits high and low affinity binding sites on cells expressing these receptors. The high affinity site is observed on cells expressing both receptors and thought to be formed by formation of a heteroreceptor complex. To study function of the heteroreceptor complex in absence of individual homoreceptor complexes we generated a heterodimer of two NGF muteins (heteromutein) such that it would not function through TrkA dimerization. Muteins Δ9/13 (9‐13aa deleted) and SS1‐NGF (7FA/H84A/R103A) form a heteromutein that, using the structural rationale, can bind only one TrkA receptor. The heteromutein should bind p75 on one side and TrkA on the other, hence be capable of forming a heteroreceptor complex. We used the heteromutein to study neurite outgrowth (TrkA function) in PC12 cells. We observe significantly higher neurite formation (55%) with the heteromutein compared to Δ9/13 (2%) and SS1‐NGF (30%) at 2 nM. The heteromutein (2 nM) also gave wild type levels of survival for PC12 cells. Our study supports a possible function for the heteroreceptor complex in augmenting neuritogenesis, and perhaps survival. (Supported by NIH grant NS24380)
Synthetic retinoid Am80 inhibits interaction of KLF5 with RAR¦Á through inducing KLF5 dephosphorylation mediated by the PI3K/Akt signaling in vascular smooth muscle cells
Authors: Zhang Xin‐hua , Zheng Bin , Han Mei , Wen Jin‐kun
Published: June 2021

In the present study, we show that Am80 inhibited the interaction between Kr¨ppel‐like factor 5 (KLF5) and RAR¦Á and this inhibitory effect was accompanied by the dephosphorylation of KLF5 in vascular smooth muscle cells (VSMCs). Treating VSMCs with LY294002, the PI3K/Akt inhibitor, abrogated Am80‐induced KLF5 dephosphorylation and reversed Am80‐induced suppression of interaction between KLF5 and RAR¦Á, whereas treating VSMCs with SB203580, the p38 kinase inhibitor, further attenuated the interaction between KLF5 and RAR¦Á. Constitutively active p38 kinase MKK6b infection prevented the KLF5 dephosphorylation induced by Am80. This study suggests that Am80 induces KLF5 dephosphorylation by activating PI3K/Akt signaling, and inhibits KLF5 phosphorylation by blocking p38 signaling, subsequently leading to the suppression of interaction of KLF5 with RAR¦Á. In conclusion£¬ we showed that Am80 significantly induces RAR¦Á expression and inhibits KLF5 expression and the interaction of KLF5 with RAR¦Á at the cellular and tissue level. Furthermore, we established that this action of Am80 is mediated by activating the PI3K/Akt pathway and inhibiting the p38 MAPK pathway. These results provide novel insight not only into the molecular mechanism by which Am80 inhibits the interaction between KLF5 and RAR¦Á but also a specific therapeutic target for vascular remodeling in diseases such as atherosclerosis.