bluuebunny/crossref_metadata_2025 huggingface dataset:
De Novo Anaplastic Ganglioglioma in a 5 Year Old Female: A Case Report
Authors: Wagner Aaron Scott , Pearl Gary S.
Published: June 2021

We report an unusual case of anaplastic ganglioglioma diagnosed do novo in a 5 year old female. The patient presented initially with new onset seizures in May of 2007. Her initial seizure workup showed a lesion of the left temporal lobe, which was subsequently 80% resected in August of 2007.

The present case is unusual for several reasons. The patient had not previously had a diagnosis of ganglioglioma and as such had not received radiation therapy. Further, some anaplastic elements demonstrated neuronal differentiation with cells staining negative for EMA, GFAP and neurofilament protein and positive for S100 and focally for synaptophysin. No reticulin positivity was seen. MIB‐1 labeling showed 60% reactivity and the tumor was felt to be most consistent with a WHO grade 4 lesion.

Anaplastic gangliogliomas (WHO grade III or IV) are rare lesions most often representing transformation or progression from previously diagnosed ganglioglioma (WHO grade I or II). Generally these lesions present following radiation therapy to affected patients. Typically the anaplastic elements demonstrate glial differentiation when immunohistochemically investigated.
Primary CNS Lymphomatoid Granulomatosis in a 62‐Year‐Old Woman with an Otherwise Unremarkable Medical History: A Case Report
Authors: Olson Ryan , Weaver Susan , Qian Jiang
Published: June 2021

Lymphomatoid granulomatosis (LYG) is a rare B‐cell lymphoproliferative disorder, strongly associated with Epstein‐Barr virus (EBV) infection. It commonly affects patients in their 4th‐6th decades of life with a 2:1 male to female ratio. Lung is the most commonly involved location, and although CNS involvement is present in 20%–30% of cases, it is rarely the primary or exclusive site. Here we report a case of LYG with isolated CNS involvement in a 62‐year‐old woman who presented with headaches and diplopia. MRI showed 3–4 small areas of punctate enhancement in the left anterior temporal lobe as well as T2 signal and FLAIR abnormalities in the surrounding temporal lobe, midbrain and right brachium pontis. Over the next 2 months she became withdrawn, less communicative and blunted in affect. Repeat MRI showed progression of the left temporal lesions and T2 signal alterations. Biopsy revealed geographic necrosis surrounded by a cellular infiltrate with markedly atypical lymphoid cells in an angiocentric pattern which were strongly positive for the B‐cell marker CD20 and EBV. Numerous CD3‐positive reactive T‐cells were present. Due to its classically angiocentric and angiodestructive scells are rare. With increased awareness of this lesion, the diagnosis can be made accurately and effective treatment initiated promptly.
Glioma‐like proliferation within tissues excised as tubers in patients with tuberous sclerosis complex: a diagnostic problem
Authors: Fischer Ingeborg , Cunliffe Clare , Bollo Robert J , Weiner Howard L , Miller Douglas C
Published: June 2021

Among 19 patients with Tuberous Sclerosis Complex (TSC), who underwent resection of tubers at our institution, five had diffuse glioma‐like infiltrates. All patients presented with refractory seizures Histologically, 4 resection specimens had the pathognomonic features of neuroglial hamartomas (tubers) and one resection specimen revealed severe cortical microdysgenesis but lacked cells typical of TSC. All lesions were associated with an infiltrate of atypical, mostly elongate, glioma‐like small cells, some of which resembled microglial cells. None of the infiltrating cells resembled reactive astrocytes, and only some of them were labelled with a GFAP immunostain. Likewise, many of the atypical cells did not label with lymphocytic or histiocytic markers. MIB‐1 (Ki67) immunostains marked a variable proportion of both microglial and glioma‐like atypical nuclei. Postoperatively (follow‐up period ranging from one to 28 months) none of the patients have any evidence of a glioma. We report here for the first time that tubers resected for treatment of seizures are sometimes associated with glioma‐like lesions, consisting of foci of hypercellularity which cannot be identified as just lympho‐histiocytic reactions nor reactive gliosis. These foci are indistinguishable from infiltrating gliomas by morphology and immunohistochemistry, and may represent a difficult diagnostic problem.
RE‐EVALUATION OF ARCHIVAL “DLDH” CASES FOR FRONTOTEMPORAL LOBAR DEGENERATION WITH UBIQUITIN INCLUSIONS: ANTIGEN RETRIEVAL IS ESSENTIAL
Authors: Gearing Marla , Cooper Deborah , Glass Jonathan
Published: June 2021

The neuropathologic subtypes of frontotemporal lobar degeneration (FTLD) can frequently be differentiated immunohistochemically. The identification of abnormal deposits of tau, ubiquitin, and, more recently, TAR DNA‐binding protein 43 (TDP‐43) and alpha‐internexin may allow separation of neuropathologic subtypes, whereas the lack of staining for these proteins may lead to a diagnosis of “dementia lacking distinctive histology” (DLDH). Because of the importance of accurate pathological diagnosis for the understanding of these disorders, we investigated whether a wider panel of antibodies and more sensitive immunohistochemical methods could allow for better neuropathologic characterization. We undertook a study of 13 autopsy cases with non‐specific diagnoses such as DLDH. We relabeled these brains with antibodies to tau, ubiquitin, TDP‐43, and alpha‐internexin. In addition, we tested the effect of antigen retrieval on the ability to detect the ubiquitin immunolabel. In 7 of the 13 cases, ubiquitin‐immunoreactive inclusions were identified. These cases also immunolabeled with the antibody to TDP‐43. This pattern of staining supported a reclassification to FTLD with ubiquitin inclusions (FTLD‐U). In all of these cases, the antigen retrieval method markedly enhanced ubiquitin immunolabel and was critical for the identification of FTLD‐U pathology. Supported by 1P50‐AG025688.
Evaluation of atopy and immune functions in children with autism spectrum disorders (ASD): Identification of an ASD subset with distinct clinical and immunological findings
Authors: Jyonouchi Harumi , Geng Lee , Cushing‐Ruby Agnes , Quraishi Huma
Published: June 2021

This study assessed prevalence of atopy, food allergy (FA), and recurrent otitis media/chronic rhinosinusitis (ROM/CRS) in 91 ASD children referred to the Allergy/Immunology Clinic along with their TLR responses (assessed by cytokine production) in comparison with non‐ASD children [ROM/CRS (N=23), FA (N=21), and healthy controls (N=48)]. Among ASD children with recurrent exacerbation of behavioral symptoms and loss of cognitive skills following infection (ASD‐RE, N=16), their TLR responses was evaluated with normal and ASD case controls. Non IgE mediated FA was highly prevalent (69.2%) in ASD children but not atopy (30.8%), ROM/CRS (14.3%), or known primary immunodeficiency (PID) (2.2%). In contrast, PID was highly prevalent in ROM/CRS children without ASD (47.8%). ASD‐RE children had a high prevalence of ROM/CRS despite absence of PID and also revealed altered production of IL‐1ß, IL‐6, and sTNFRII with TLR7/8 agonist with or without LPS pretreatment. They also revealed altered IL‐10 production with TLR2/6 agonist. Thus ASD‐RE children revealed distinct clinical features and laboratory findings not associated with atopy or PID. Their aberrant TLR 7/8 and 2/6 responses may be associated with recurrent infection and possibly impaired stress responses, given roles of these cytokines in neuro‐immune network. Funded by Jonty Foundation/ARI.
Adult Myopathies with Tubular Aggregates: Clinical Correlates
Authors: Gultekin Sakir Humayun , Grafe Marjorie , Cupler Edward
Published: June 2021

Tubular aggregates (TA) are rare findings in muscle biopsies. They have been associated with periodic paralyses and myotonia, and other sporadic and familial myopathies. They may originate from sarcoplasmic reticulum, T‐ tubules or mitochondrial membranes. We report 3 patients with TA as the predominant abnormality in their muscle biopsy. The age range was 30–63. The complaints included progressive single extremity weakness, weak legs with respiratory difficulty, and muscle stiffness. 1 patient had a family history of muscle cramps, and another patient was a renal transplant recipient on multiple immunosuppressants. Neurological examination revealed focal or diffuse mild extremity weakness. EMG in 2 patients showed myopathy. The CPK values were normal to mildly elevated. Muscle biopsies of all three patients revealed focal or diffuse TA in Type II fibers by NADH stain, 2 with electron microscopic confirmation. There was no inflammation, necrotizing myopathy, dystrophic changes, mitochondrial changes, or abnormal metabolic deposits with histochemical, immunohistochemical, and ultrastructural studies. Myopathy with tubular aggregates should be considered in the differential diagnosis of metabolic and toxic myopathies and may be observed in a broad age range of adult patients with myopathy. The focal extremity weakness may lead to an initial consideration of radiculopathy.
CD24 inhibits demyelination in a mouse cuprizone‐demyelination model
Authors: Ray‐Chaudhury Abhik , Carl Joseph W
Published: June 2021

CD24 is a glycosyl‐phosphatidylinositol (GPI) anchored protein expressed in the developing central nervous system (CNS). In adult CNS, CD24 is mainly expressed on ependymal cells and on neurons in zones of neurongenesis. However, its function in the CNS remains unclear. We have previously shown CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE). Mice deficient for CD24 are resistant to the induction of EAE, on the other hand, mice over‐expressing CD24 on astrocytes had a more severe course of disease with more de‐myelination after EAE induction. To test whether CD24 is involved in demyelination/remyelination process, we used a cuprizone model of demyelination to study the role of CD24 in demyelination/remyelination. At week 4 after cuprizone diet, the CD24‐KO mice had significantly less de‐myelination compared to WT mice, but by week 6 demyelination levels were the same. CD24‐KO mice remyelinated the corpus collosum at the same rate as WT mice after returning them to a normal diet following 6 weeks of exposure to 0.2% cuprizone diet. This study indicates that CD24 does not play a major role in the re‐myelination process, but inhibits the de‐myelination process.

Supported by a Davis/Bremer Medical Research Grant, The Ohio State University, Office of Research, College of Medicine, Columbus Oh, 43210
Role of nitric oxide (NO) in the hyperbaric oxygen (HBO2)‐induced long‐lasting activation of endogenous opioid systems in mice
Authors: Zylstra Carlyn C , Zelinski Lisa M , Chung Eunhee , Ohgami Yusuke , Shirachi Donald Y , Quock Raymond M
Published: June 2021

Exposure to HBO2 causes a long‐lasting analgesic effect in patients with chronic pain (Kiralp et al., J. Int. Med. Res. 32:258–262, 2004). To determine the duration of antinociceptive effect in an animal model following exposure to HBO2, male NIH Swiss mice (20–30 g) were exposed to HBO2 at 2.5 ATA for 60 min then removed to room air for varying durations of time (5, 15, 30, 60, 90, 120, 150 or 180 min) prior to antinociceptive testing. The antinociceptive effect of HBO2 remained robust up to 90 min but subsided by 120 min. By contrast, mice exposed to 70% nitrous oxide for 60 min exhibited antinociception only through the 15‐min interval. When administered prior to HBO2 exposure, both naltrexone and L‐NAME antagonized the antinociceptive effect seen 90 min later. When administered prior to antinociceptive testing, naltrexone but not L‐NAME antagonized the antinociceptive effect of HBO2. These results suggest that there is a critical role of NO during HBO2 exposure that is required for expression of the opioid‐mediated antinociceptive effect 90 min later. (Supported in part by NIH Grant GM‐77153, ASPET SURF Program and the Chico Hyperbaric Center).
Insulin regulation of the human dopamine transporter activity: A GSK‐3 dependent pathway
Authors: Speed Nicole , Saunders Christine , Galli Aurelio
Published: June 2021

The dopamine transporter (DAT) is the primary pathway for the clearance of extracellular dopamine (DA), and thus determines the magnitude and duration of the dopaminergic signaling. We have shown that insulin, and subsequent signaling through PI3‐kinase dependent pathway, is able to regulate DAT cell surface expression. This trafficking phenomenon is mediated by Akt, a kinase which inhibits glycogen synthase kinase‐3 (GSK‐3). Here, we use human embryonic kidney cells stably expressing human DAT (hDAT cells) to investigate downstream cellular components important in this Akt effect. Our data suggest that SB 216763, an inhibitor of GSK‐3, increases DAT cell surface expression in both hDAT cells and straiatal synaptosomes Therefore, our data demonstrate that DAT cell surface expression is regulated by insulin and that Akt, through GSK‐3, plays a key role in the regulation of trafficking of the DAT.
An allosteric potentiator suggests a role for M4 muscarinic acetylcholine receptor (mAChR) in modulating excitatory hippocampal synaptic transmission
Authors: Shirey Jana K , Xiang Zixiu , Orton Darren , Brady Ashley E , Johnson Kari A , Williams Richard , Ayala Jennifer E , Rodriguez Alice L , Wess Jurgen , Weaver David , Niswender Colleen M , Conn P Jeffrey
Published: June 2021

Numerous animal and human studies suggest that muscarinic acetylcholine receptors (mAChRs), specifically M1 and M4, may be viable targets for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). We have used cheminformatic and medicinal chemistry to develop novel, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine (ACh) 47‐fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for ACh and coupling to G proteins. Whole cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increased carbachol‐induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout (KO) mice. This provides compelling evidence for selective regulation of excitatory transmission in this important forebrain structure by M4. We are now examining the effects of selective M1 and M4 activation on field excitatory postsynaptic potentials in the CA1 region to understand the involvement of these receptors in muscarinic long‐term potentiation and depression.

This work supported by NIMH grants MH073676‐01A1 and 1 F31 MH80559‐01.
Synthesis of New 4‐Benzothiazolylamino‐6‐methyl pyran‐2‐ones and their Antibacterial Activity
Authors: Hoti Ramiz , Kalaj Vuksan , Bicaj Muhamet , Mulliqi‐Osmani Gjyle , Vehapi Idriz , Ismaili Hamit
Published: June 2021

4‐Benzothiazolylamino‐6‐methyl‐2H[1]‐pyran‐2‐one 6a , 4‐(4‐Methylbenzothiazolylamino)‐6‐methyl‐2H[1]‐pyran‐2‐one 6b , 4‐(5,6‐dimethylbenzothiazolylamino)‐6‐methyl‐2H[1]‐pyran‐2‐one 6c and substituted 4‐(Benzothiazolylamino)‐6‐methyl‐3‐nitro‐2H[1]‐pyran‐2‐ones 6(d, e) are synthesized by condensation of 4‐Chlor‐6 methylpyran‐2‐ones (2, 4) and corresponding benzithiazolylamines 5(a‐e) under reflux reaction conditions. The antibacterial activity of products 4(a‐d) against Staphylococcus aureus, Clebsiella and Escherichia coli were investigated. Compounds 6(a‐e) showed light bactericide activity against Staphylococcus aureus, Escherichia coli and Clebsiella. Compounds 6e and 6c were more active against Staphylococcus aureus. Compounds 6d and 6b exhibited the strongest activity against Clebsiella, whereas 6e was more active against Escherichia coli. In general there was a driect positive correlation between increasing concentration of the compound and anitbacterial activity.
Defect and Rescue of NAADP‐activated Ca2+ Channel Activity in Mucolipin‐1 Deficient Human Fibroblasts
Authors: Zhang Fan , Jin Si , Yi Fan , Thomas Cindy , Li Pin‐Lan
Published: June 2021

Recent studies have indicated that mucolipin‐1 (TRP‐ML1) may be a lysosomal Ca2+ channel activated by a novel Ca2+signaling nucleotide, NAADP. The present study attempted to further test the hypothesis that NAADP functions as an endogenous activator of TRP‐ML1 using human fibroblasts. By reconstituting lysosomal channels, we found that lysosomes from TRP‐ML−/− cells had much lower Ca2+ channel activity (NPO=0.00406 ±0.0009) compared to TRP‐ML+/+ cells (NPO=0.00562 ±0.00176). NAADP (100 nM) significantly increased the activity of lysosomal Ca2+ channel from TRP‐ML1+/+ cells (NPO =0.0547±0.0105), but it had no effect on these channels from TRP‐ML1−/− cells. When TRP‐ML−/−cells was transfected with TRP‐ML1 gene, the channel activity was restored. Fluorescence imaging showed, ET‐1, a potent stimulator of NAADP production activated lysosome‐related local Ca2+ burst followed by global Ca2+ release in TRP‐ML1+/+ cells, but not in TRP‐ML1−/ − cells. In TRP‐ML1 transfected TRP‐ML1−/− cells, ET‐1‐induced Ca2+ response was rescued. Similarly, confocal microscopy also demonstrated that ET‐1 induced local Ca2+ release from lysosomes in TRP‐ML1+/+ or gene rescued TRP‐ML1−/−fibroblasts, rather than TRP‐ML1−/− cells. All these results indicate that NAADP may target TRP‐ML1 to release Ca2+ from lysosomes and participates in NAADP‐mediated signaling regulation (Supported by NIH grants HL057244 and HL075316).
Regulation of KCNQ5 potassium channels in A7r5 vascular smooth muscle cells involves protein kinase C‐mediated channel phosphorylation
Authors: Olsen Jessica L , Brueggemann Lioubov I , Cribbs Leanne L , Byron Kenneth L
Published: June 2021

Protein kinase C (PKC) ‐dependent suppression of KCNQ5 voltage‐dependent K+ currents was recently implicated in signal transduction of physiological concentrations of arginine vasopressin (AVP). It has yet to be determined if KCNQ5 channel proteins are directly phosphorylated in response to PKC activation. A FLAG‐ and GFP‐tagged KCNQ5 (FLAG‐KCNQ5) construct was designed and transfected into A7r5 cells. Whole‐cell voltage clamp techniques were used to confirm the expression of functional FLAG‐KCNQ5 channels. FLAG‐KCNQ5 currents were 100‐fold larger than endogenous currents and the currents were abolished by expression of KCNQ5 shRNA. AVP (500 pM) and PKC activator 4β‐phorbol 12‐myristate 13‐acetate (PMA, 1 nM) reduced FLAG‐KCNQ5 currents by about 80%. FLAG‐KCNQ5 channel protein was detected as a band at 105 KDa in anti‐FLAG immunoprecipitates by Western blotting. The same band was recognized by anti‐ phospho‐serine/threonine/tyrosine antibodies following treatment with PMA (1 nM, 15 min). Phosphorylation was abolished by preincubation with the PKC inhibitor calphostin C (250 nM, 2hrs). A‐kinase anchoring protein (AKAP150), which has been implicated in recruitment of protein kinases and channel phosphorylation in other cell systems, was found to co‐immunoprecipitate with FLAG‐KCNQ5 channels. We conclude that KCNQ5 channel proteins are directly phosphorylated in response to PKC activation in A7r5 cells. Supported by NIH R01 HL070670 (KLB) and a 2007 Student Scholarship in Cardiovascular Disease and Stroke (JLO).
Characterization of ClC‐2 Chloride Channel/PKA Interaction in Mouse Heart
Authors: Huang Zheng Maggie , Britton Fiona , Yuan Can , An Changlong , Hatton William , Duan Dayue
Published: June 2021

Background: A ClC‐2 encoded chloride inward rectifier channel (Cl.ir) has been identified in the heart of several species. Cl.ir may play a role in regulation of pacemaker activity of the heart. In this study, we studied the regulation of mouse cardiac ClC‐2 channels by protein kinase A (PKA) phosphorylation.

Methods: RT‐PCR and Western Blot were used to examine the distribution of mClC‐2 in mouse heart. Whole‐cell voltage‐clamp was used to study the regulation mClC‐2 in NIH/3T3 cells. Coimmunoprecipitation (CoIP) and yeast two hybrid (Y2H) were used to identify the binding partners of mClC‐2 in the heart.

Results: 1). ClC‐2 exists in mouse heart. Analysis showed 765Ser as a PKA phosphorylation site. 2). Expression of ClC‐2 in NIH/3T3 cells generated functional channels. Treatment by 8‐Br‐cAMP to activate PKA increased current density from −16.2±2.8 to −20.6±3.5 pA/pF at −140mV by 27.4±5% (mean±SD, n=3). The increase was reversed to −15.5±3.3pA/pF by 26.6±4.1% upon PKA inhibition. Phosphotases PP1 and PP2 inhibitor Calyculin A further increased current by 22±3.1%. 3). CoIP using mouse heart and mouse atrial cell line HL1 lysates both showed association of ClC‐2 to 1 adrenergic receptors, PKA catalytic subunit, and PP1. 4). Y2H identified PP1 as a binding partner of ClC‐2.

Conclusion: Mouse cardiac ClC‐2 channels may be regulated by phosphorylation/dephosphorylation through a direct interaction with PKA and PP1.

Source of support: National Institutes of Health, National Center for Research Resources, National Heart, Lung, and Blood Institute and American Heart Association Western Affiliate.
No relationship found between RNA editing of the serotonin 2c receptor and expression of Gαq protein
Authors: Canal Clinton Ernest , Mahautmr Kontip Connie , Jacobs Michelle M. , Sanders‐Bush Elaine , Airey David C.
Published: June 2021

RNA editing of transcripts of the serotonin 2c receptor (5HT2CR) occurs at up to five places within the putative second intracellular loop of the receptor, a region involved in modulating receptor coupling to Gαq proteins. 5HT2CR edited isoforms differ in signal transduction efficiency depending on the degree of editing. In vitro, the fully edited (VGV) 5HT2CR shows the lowest level of constitutive and agonist‐stimulated activity, whereas the nonedited (INI) 5HT2CR exhibits the converse pattern. The mechanisms underlying the changes in signal transduction related to RNA editing of 5HT2CRs may involve changes in Gαq protein levels. To test the causality of a 5HT2CR‐Gαq relation, we measured RNA editing of 5HT2CR in Gαq knock‐out (KO) mice and Gq protein levels in transgenic mice solely expressing the INI isoform of 5HT2CR. Pyrosequences of RNA isolated from amygdaloid cortex of Gαq KO and wild‐type (WT) mice revealed no significant differences in 5HT2CR mRNA editing profiles. Cortical tissue from INI and WT mice was assayed separately for expression of Gαq and Gα11 subunits by Western blotting. No differences in signal density between INI and WT groups were found, suggesting equivalent levels of Gαq and Gα11 protein. Together, the data do not support a causal or compensatory relation between 5HT2CR RNA editing and Gq protein levels.

Supported by: NIH T32 MH065215 (CEC), R01‐MH34007 (ESB) and R21‐MH077942 (DCA)
Possible role of Angiotensin II Receptor (AT2) in regulation of Insulin Receptor signaling
Authors: Ramdas Maya , Kolhe Ravindra , Gavini Nara , Pulakat Lakshmi
Published: June 2021

Renin angiotensin system plays an important role in the development of metabolic syndrome, primarily characterized by insulin resistance, which poses a potential risk for other factors like obesity, hypertension etc. The actions of angiotensin II mediated through the AT1 receptor are well studied, but the role of the AT2 receptor is not clearly established. We have shown that the AT2 can interact with the beta subunit of Insulin Receptor (IR) in yeast two‐hybrid assay. To further evaluate the effects of AT2 on the signaling mechanism of IR, we studied the interaction between the two proteins in Chinese Hamster Ovary (CHO) cells and the human breast cancer (MCF‐7) cells. In our studies using CHO cells, we showed that transient transfection of the AT2 could result in AT2‐IR complex formation and lack of phosphorylation of IR‐beta. However, a truncated AT2 in which C‐terminal cytoplasmic region was deleted did not show interaction with the IR‐beta. MCF‐7 cells constitutively express the AT1 receptor. We have seen that starving these cells for 6 hours or more changes the expression pattern and the levels of the AT1 goes down while the levels of the AT2 increases. These cells expressing the AT2 receptor show lack of phosphorylation of the IR‐beta. Based on these data, we plan to study the proteomics in these cells to further understand the cross talk between angiotensin II and insulin signaling at a cellular and molecular level.
Binding and functional characterisation of allosteric agonists at M2 muscarinic acetylcholine receptors
Authors: Gregory Karen Joan , Hall Nathan E. , Sexton Patrick M. , Christopoulos Arthur
Published: June 2021

Muscarinic acetylcholine receptors (mAChRs) contain at least one allosteric site that is topographically distinct from the acetylcholine (ACh)‐binding orthosteric site. Although numerous studies have investigated the structural basis of allosteric modulation at these receptors, far less is known about allosteric ligands that activate the receptor in their own right. We generated a number of M2 mAChRs harboring different orthosteric‐site mutations (W99A, Y403A), or allosteric‐site mutations (172EDGE175‐QNGQ, Y177A), and investigated their impact on the binding and function of the putative allosteric agonists AC‐42, 77‐LH‐28‐1, McN‐A‐343 and N‐desmethylclozapine (NDMC). As expected, orthosteric site mutations reduced the affinity and/or efficacy of the orthosteric agonists ACh and pilocarpine, whereas the allosteric site mutations had no significant effects. W99A and Y403A caused significant increases in the affinity (100–300 fold and 3–10 fold respectively) and efficacy of AC‐42 and 77‐LH‐28‐1. NDMC and McN‐A‐343 showed increased efficacy, but not affinity, at Y403A and Y177A. Homology models of the M2 mAChR have been developed around the different ligands. The experimental data and modelling together lead to a better understanding of the allosteric binding sites of mAChRs. This work is supported by National Health & Medical Research Council Project Grant No. 400134 and the Dowd Foundation.
Zyxin is Involved in Thrombin Signaling via Interaction with PAR‐1 Receptor
Authors: Voyno‐Yasenetskaya Tatyana , Han Jingyan
Published: June 2021

Thrombin‐induced increase in endothelial monolayer permeability depends on reorganization of the cytoskeletal structures. Protease‐activated receptor‐1 (PAR‐1) is the main receptor responsible for thrombin signaling in endothelial cells. Here we showed that PAR‐1 receptor interacted with zyxin, a LIM domain containing phosphoprotein, both in vitro and in vivo. We have shown that LIM domain 1 and 2 of zyxin interacted with C‐terminal domain of PAR‐1 receptor. In human umbilical vein endothelial cells (HUVECs), thrombin induced recruitment of the endogenous zyxin to the plasma membrane, promoted zyxin targeting to the focal adhesion, and promoted zyxin association with the actin stress fibers. Downregulation of zyxin using siRNA technique inhibited thrombin‐induced recruitment of FAK to focal adhesions. Similarly, disruption of the interaction between endogenous zyxin of PAR‐1 receptor by overexpression of the C‐terminal domain of zyxin inhibited thrombin‐induced recruitment of FAK to focal adhesions. In addition, downregulation of zyxin using siRNA resulted in reduced cellular responses to thrombin stimulation, such as cell rounding, actin stress fiber formation and serum response factor‐dependent gene expression. These results suggest that zyxin is involved in thrombin signaling by direct interaction with PAR‐1 receptor.
Localization of the Dopamine D2 Receptor in Lipid Raft Microdomains: Implications for Receptor Function
Authors: Romanelli Robert John , Neve Kim
Published: June 2021

The dopamine receptors are a subclass of G protein‐coupled receptors (GPCRs), including the D1‐like and D2‐like receptors, which have been implicated in working memory, motivation/reward, and motor activity. Antagonists to the D2‐like receptors inhibit the psychotic symptoms associated with schizophrenia and block the motor stimulant and rewarding properties of drugs of abuse. Accordingly, the molecular underpinnings of signaling through the D2‐like receptors are of great clinical interest. In the present study we sought to investigate the role of lipid raft microdomains in D2 receptor (D2R) function. Lipid rafts are regions of the plasma membrane enriched in glycosphingolipids and cholesterol, and have been implicated widely in the regulation of GPCR signaling. We demonstrate through sucrose gradient fractionation and indirect immunofluorescence that D2Rs co‐localize with markers for lipid rafts in HEK293 cells. We also show that endogenous D2R from striatal membranes localize within lipid rafts. We report that depletion of cholesterol with methyl‐beta‐cyclodextrin (MCD), an agent well characterized to disrupt lipid rafts, significantly impairs D2R‐mediated inhibition of cAMP production in HEK293 cells. The results of these studies demonstrate that the localization of D2Rs in lipid rafts regulates D2R‐induced activation of its cognate G proteins.
Heparin Binding‐Epidermal Growth Factor and Src Family Kinases in Proliferation of Renal Epithelial Cells
Authors: Zhuang Shougang , Kinsey Gilbert R. , Rasbach Kyle
Published: June 2021

This study was to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate. RPTC expressed both pro‐ and cleaved heparin binding epidermal growth factor (HB‐EGF) forms and several metalloproteinases (MMP‐2, ‐3 ‐9, and ADAM10, ADAM17) that have been reported to cleave HB‐EGF. Treatment of RPTC with CRM 197, an inhibitor of HB‐EGF binding to EGF receptor, or down‐regulation of HB‐EGF with siRNA inhibited RPTC proliferation following plating. Furthermore, blocking metalloproteinases with GM6001, TAPI‐1, GW280264X, but not GI254023X, attenuated the proliferation following plating. Although EGF receptor activation is required for RPTC proliferation following oxidant injury, CRM197, GM6001, and TAPI‐1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation in oxidant‐injured RPTC. In addition, PP1 treatment attenuated HB‐EGF‐enhanced RPTC proliferation. We suggest that RPTC proliferation following plating is regulated by HB‐EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB‐EGF production.
Lysophosphatidic acid receptor and Ral signaling in breast cancer cell migration and invasion
Authors: Li Timothy Tsz‐Tim , Au‐Yeung Catherine , Alemayehu Mistre , Pape Cynthia , Pampillo Macarena , Laird Dale , Babwah Andy V , Bhattacharya Moshmi
Published: June 2021

Lysophosphatidic acid (LPA) is a simple lipid molecule that mediates a variety of processes such as cell survival and growth, and plays a role in the progression of a variety of cancers, stimulating cell proliferation, migration and invasion. The effects of LPA are mediated via the activity of its G protein‐coupled receptors, LPA1‐3. Recently, LPA has been shown to enhance metastasis of breast cancer to bone. However, the mechanisms by which LPA receptor signaling regulates cell migration and invasion of breast cancer cells remains unclear. Breast cancer cell proliferation has been shown to be stimulated by the small G protein Ral. Ral activity can be regulated by its guanine‐nucleotide exchange factors (RalGEFs) and the multifunctional protein β‐arrestin. We compared expression of LPA receptors, Ral, RalGDS, and β‐arrestin in the non‐tumorigenic mammary cell line MCF‐10A and the invasive breast cancer cell lines MDA‐MB‐435 and MDA‐MB‐231. Our data shows for the first time that both breast cancer cell lines have higher expression of β‐arrestin compared to MCF‐10A cells. Furthermore, MDA‐MB‐231, but not MCF‐10A cells migrate and invade in response to LPA via pertussis toxin‐sensitive G proteins and via a β‐arrestin‐Ral pathway. In summary, our data demonstrates a novel mechanism by which LPA receptors may regulate breast cancer cell migration and invasion.

Research is funded by CIHR awarded to Dr. M Bhattachary
Treatment with the complement inhibitor, FUT‐175 decreases PMN activation and myocardial infarct size in Sprague‐Dawley rats
Authors: Bonte Laura R La , Davis‐Gorman Grace , Stahl Gregory L , McDonagh Paul F
Published: June 2021

Complement activation plays an important role in the inflammatory response and is involved in ischemia/reperfusion (I/R) injury. The purpose of this study was to determine if the complement inhibitor, FUT175, attenuates neutrophil‐mediated myocardial I/R injury. Sprague‐Dawley (SD) rats underwent 30min of coronary artery occlusion followed by 120min of reperfusion. Treated SD rats were given FUT175 (1mg/kg) i.v. 5min prior to reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition following reperfusion. We found that there was significantly increased complement deposition in the untreated SD LV compared to the FUT175 treated SD LV (43.9±6.1 %C3/LV vs. 14.7±6.0 %C3/LV, p<0.05). We also found that the infarct size of the FUT175 treated SD rats was significantly decreased (49.2±5.1 %AI/AAR vs. 21.1±4.8 %AI/AAR, p<0.01). Whole blood samples were taken pre‐ischemia, after 15min of reperfusion, and after 120min of reperfusion. Blood samples were processed for flow cytometric analysis of CD11b expression on activated PMNs at each time point. PMNs from the FUT175 treated rats exhibited significantly decreased CD11b expression compared to the untreated group (p<0.05). Taken together, these findings indicate a role for complement in the severity of I/R injury and that this injury can be attenuated with the complement inhibitor FUT175. Supported by NIH 58859 and AHA 0610018Z.
Ischemic Myocardial Protection In Transgenic Mice With Cardiac α1A‐Adrenergic Receptor Overexpression
Authors: Zhao Xin , Zhang Yan , Shen You‐Tang , Yan Lin , Vatner Dorothy E. , Graham Robert M. , Vatner Stephen F.
Published: June 2021

It is thought that activation of α1A‐adrenergic receptor (α1A‐AR) signaling may be involved in myocardial ischemic preconditioning(IP). If this is correct, then a transgenic (TG) model in which the α1A‐AR is specifically overexpressed only in the heart should be useful for exploring IP mechanisms. To determine if TG mice with cardiac specific overexpression of the α1A ‐AR (66‐fold) exhibited IP, TG mice and their non‐transgenic (NTG) littermates were subjected to left anterior descending coronary artery occlusion for 60 min followed by reperfusion (n=5 in each group). After 24hr, the hearts were perfused and the area at risk determined by Alcian Blue, and infarct area determined by 2, 3, 5‐triphenyltetrazolium chloride (TTC) staining. There was no difference in the area at risk between the TG and NTG groups. However, infarct size, expressed by a fraction of area at risk was significantly lower, p<0.01, in the TG group (31±3.4%) compared to the NTG animals (52±3.6%). The frequency of apoptosis (TUNEL) in the area at risk in TG mice was also significant reduced, p<0.05, compared to NTG mice (0.53±0.2 vs. 1.60±0.5 myocytes/field). Thus, the TG α1A‐AR mice exhibited marked protection against the development of infarction and apoptosis, indicating that this model should be uniquely useful for understanding the role of α1A‐AR in IP.
Microvascular Network Restructuring Associated with MMP Inhibition in Spontaneously Hypertensive Rats
Authors: Chen Andrew , DeLano Frank A. , Schmid‐Schönbein Geert W. , Murfee Walter L.
Published: June 2021

Recently, we have shown that MMP inhibition via doxcycyline treatment normalizes blood pressure and attenuates cell dysfunction, i.e. insulin resistance, in adult spontaneously hypertensive (SHR) rats. The objective of this work was to determine whether MMP inhibition leading to blood pressure normalization in hypertensive animals is associated with microvascular network structural alterations. Age matched (14–16 wks) SHR and Wistar‐Kyoto (WKY) rats were administered normal drinking water with and without doxycycline (5.4 mg/kg/d) for 12 weeks. Mesenteric tissues were harvested from 3–5 animals per experimental group and immunolabeled for CD31. Intact microvascular networks were compared based on 3 metrics: vascular area per total tissue area, vessel length per vascular area, and capillary sprouts (blind ended vessels) per vascular area. Microvascular networks from control SHR animals exhibited decreased vascular area per tissue area (P=0.030) and increased vessel length per vascular area (P=0.039) compared to the control WKY group. Networks from the doxycycline treated‐SHR group did not exhibit similar differences. Additionally, doxycylcline treatment served to decrease capillary sprouting per vascular area in SHR animals (P=0.039). These results suggest that microvascular network patterning is linked to MMP activity in adult spontaneously hypertensive rats. Supported by NIH‐HL10881.
The time‐dependency of post‐conditioning for neuroprotection against global cerebral ischemia and reperfusion injury in C57BL/6 mice
Authors: SHEN Jia , SUN Lina , LIAO Yuanjin , LIN Shupeng , BAI Xiaoyin , XIA Qiang
Published: June 2021

To establish (a) suitable treatment protocol(s) for neuroprotection in mice, the post‐conditioning treatments were performed by 3 cycles of reperfusion and occlusion (5s/5s, 10s/10s, 15s/15s, 30s/30s and 45s/45s, respectively) on bilateral common carotid artery immediately after 10 min bilateral common carotid artery occlusion (BCCAO) in male C57BL/6 mice. The escape latency was the shortest in Morris water maze test and the recovery of cerebral blood flow after reperfusion was complete in 15s/15s post‐conditioning group while the mice in 30s/30s group performed best in beam walking test. Calcium overload as well as neuron death rate were depressed most significantly in hippocampus and striatum in 15s/15s group and 30s/30s group respectively. Additionally, we tested the neuroprotective efficiency of 15s/15s postconditioning applied at 5min, 15min, 30min or 1h after the reperfusion and found that the protective effect was decreasing when increasing the delay time of postconditioning. Delay for more than 30min eliminated the protective effect of post‐conditioning. We concluded that appropriate post‐conditioning (15s/15s and 30s/30s, 3 cycles) provides better neuroprotection against global cerebral ischemia and reperfusion injury in mice.
Heme oxygenase‐1 in post‐ischemic reperfusion brain damage: effect of cyanidin‐3‐O‐beta‐glucoside
Authors: Di Giacomo Claudia , Acquaviva Rosaria , Sorrenti Valeria , Galvano Fabio , Vanella Luca , Volti Giovanni Li , Vanella Angelo
Published: June 2021

Cerebral ischemia is the result of decreased supply of blood to the brain or a part of it. The consequent neuronal injury arises from a complex pattern of cellular reactions, including oxidative stress. Heme oxygenase‐1 (HO‐1) is an inducible stress protein that provides cytoprotection against oxidative stress. Recently it has been reported that polyphenolic natural compounds are able to induce potently HO‐1 expression, exerciting protective effects. Cyanidin‐3‐O‐beta‐D‐glucoside (C3G) is a natural compound possessing anti‐oxidant and anti‐inflammatory properties. This study investigated the effect of C3G on an experimental model of partial and transient cerebral ischemia in the rats. Several cellular markers of oxidative stress were evaluated in the brains of rats submitted to 20 min cerebral ischemia followed by different times of post‐ischemic reperfusion. In the same experimental conditions HO‐1 expression was also evaluated by western blot analysis. Results obtained demonstrated that C3G treatment is able to protect brain tissue from post‐ischemic reperfusion damage and suggested that these protective effects are partially due to an induction of HO‐1.
Polychlorinated biphenyls upregulate P‐glycoprotein expression in brain microvascular endothelial cells
Authors: Radakovic Masa , Eum Sung Yong , Couraud Pierre‐Olivier , Hennig Bernhard , Toborek Michal
Published: June 2021

Blood‐brain barrier (BBB) is composed of capillary endothelium expressing low paracellular permeability and potent, multispecific drug‐efflux pumps, such as P‐glycoprotein (P‐gp). As a primary gatekeeper of the BBB, P‐gp acts as a major impediment to central nervous system (CNS) pharmacotherapy. Physiological regulation of this xenobiotic transporter is affected by a variety of environmental stimuli. Polychlorinated biphenyls (PCBs) are an example of such environmental toxicants, known to induce neurotoxicity and a range of other harmful health effects. Here we investigate the effects of specific PCB congeners on expression and activity of P‐gp. Treatment of human brain endothelial cells (HBMEC) with PCB 126 and 153 dose‐dependently increased P‐gp protein levels. PCB‐mediated changes in P‐gp expression correlated with increased rhodamine 123 efflux (R 123), indicating the up‐regulation of transporter functions of P‐gp. R 123 accumulation was modulated by a variety of P‐gp inhibitors, which exhibited different rates of effectiveness. These results suggest that PCB‐induced overexpression of P‐gp in brain microvessels may reduce the efficacy of pharmacological treatments of the CNS disorders in PCB‐exposed population. In additon, application of P‐gp blockers may improve the adverse effects of PCBs in the CNS.
Homocysteine attenuates blood brain barrier function by inducing oxidative stress and the junctional proteins
Authors: Tyagi Neetu , Kumar Munish , Pushpakumar SB , Lominadze David , Moshal Karni S , Sen Utpal , Vacek Thomas Paul , Tyagi Suresh C
Published: June 2021

Hyperhomocysteinmia (HHcy) is associated with neurological disorders and causes blood brain barrier (BBB) dysfunction. However, the mechanism of BBB dysfunction is unclear. We hypothesize that Hcy induces oxidative stress, activates inter‐endothelial junctional (TJ) proteins leading to BBB dysfunction. In this study mosue brain microvascular endothelial cells (MBEC) were treated with different doses of homocysteine (Hcy) for 18hrs. Reactive oxygen species (ROS) was detected using DCFH‐DA assay. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was analysised by Western‐blotting. The interaction of junctional proteins were investigated using co‐immunoprecipitation. Our results show that Hcy induces oxidative stress, which causes down regulation of the inter‐endothelial junctional proteins ((ZO‐1, caludin, occuldin). Furthermore, oxidative stress leads to disruption of the cadherin‐beta‐catenin complex and an activation of extracellular signal‐regulated kinase. In conclusion, Hcy‐induced oxidative stress leads to BBB dysfunction, in part by, activating TJ proteins.
Involvement of the hypothalamic paraventricular nucleus on the cardiovascular responses to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats
Authors: Crestani Carlos Cesar , Tavares Rodrigo Fiacori , Busnardo Cristiane , Alves Fernando Henrique Ferrari , Correa Fernando Morgan Aguiar
Published: June 2021

It was previously demonstrated that noradrenaline (NA) microinjected into the bed nucleus of the stria terminalis (BST) evokes pressor and bradycardiac responses, which are mediated by acute systemic vasopressin release. The aim of the present study was to evaluate the involvement of the paraventricular (PVN) or supraoptic (SON) hypothalamic nuclei that synthesize vasopressin, on the cardiovascular responses to NA microinjected into the BST of unanesthetized rats.

For this purpose, male Wistar rats had guide cannulas implanted in the BST and ipsilaterally in the PVN or SON and a polyethylene catheters was implanted into the femoral artery for mean arterial pressure (MAP) and heart rate (HR) recording.

PVN pretreatment with the nonselective synapse blocker CoCl2 (1mM) significantly reduced the pressor (32±4 vs 7±2 mmHg; t=6, p<0.05) and bradycardiac (−39±7 vs −6±2 bpm; t=7, p<0.05) responses to NA microinjected into the BST when compared to the response before PVN treatment. However, CoCl2 microinjected into the SON previously to NA microinjection into the BST did not affect the pressor (30±3 vs 29±2 mmHg; t=0.2, p>0.05) and bradycardiac (−34±3 vs −34±5 bpm; t=0.1, p>0.05) response.

Thus, our results suggest that the PVN but not the SON is involved in the mediation of the cardiovascular responses to NA microinjected into the BST

Research support: FAPESP and FAEPA
Central injections of adeno‐associated virus‐siRNA gene to silence estrogen receptor beta (ERβ) augment aldosterone‐induced hypertension in female rats
Authors: Xue Baojian , Badaue‐Passos Daniel , Beltz Terry G , Johnson Ralph F , Johnson Alan Kim , Hay Meredith
Published: June 2021

Previous studies have shown that ovariectomy or central administration of a non‐selective estrogen receptor (ER) antagonist augments aldosterone (ALDO)‐induced hypertension in female rats. The present study further investigated which ER subtype is responsible for the protective effects of estrogen on ALDO‐induced hypertension. Aortic blood pressure was measured in female rats with the use of telemetry implants. ALDO (750 ng/h) was administered subcutaneously via an osmotic pump. At the same time, central injections of recombinant adeno‐associated virus (AAV) carrying small interference (si) RNA silencers of ERα (ERα‐siRNA), ERβ (ERβ‐siRNA) or scrambled siRNA (SCM‐siRNA) were used to knock down central ERα and ERβ subtypes. Both intracerebroventricular (icv) and paraventricular nucleus injections of AAV‐ERβ‐siRNA augmented ALDO‐induced hypertension (Δ25.4±0.6 mmHg, n=2 and Δ15.8±0.4 mmHg, n=3, respectively). However, rats with icv injections of ERα‐siRNA did not increase blood pressure induced by ALDO (Δ8.4±3.0 mmHg, n=2) in comparison to SCM‐siRNA rats (Δ9.2±2.2 mmHg, n=3). These data indicate that central ERβ mediates the protective effects of estrogen against ALDO‐induced hypertension. (Support: NIH HL‐59676 and HL‐62261)
Intracerebroventricular injections of adeno‐associated virus‐siRNA gene to silence angiotensin type 1 receptors (AT1) prevent aldosterone‐induced hypertension
Authors: Xue Baojian , Badaue‐Passos Daniel , Beltz Terry G , Johnson Ralph F , Johnson Alan Kim , Hay Meredith
Published: June 2021

Many studies have implicated aldosterone (ALDO) in the progression of renal injury, cardiac remodeling after myocardial infarction, and the development of hypertension. In several cases, these effects of ALDO appear to involve an interaction with the renin‐angiotensin system (RAS). In the present study, we investigated the effect of intracerebroventricular (ICV) injections of recombinant adeno‐associated virus (AAV) carrying small interference (si) RNA silencers of angiotensin type 1 receptors (AT1‐siRNA) or scrambled siRNA (SCM‐siRNA) on ALDO‐induced hypertension in male rats. AAV (2 μ1) was injected ICV at the same time systemic ALDO infusion (750 ng/h) was started. Arterial blood pressure was continuously measured by telemetry. ICV AAV‐AT1‐siRNA injection attenuated the development of ALDO‐induced hypertension (104.3±1.4 to 120.6±1.8 mmHg in SCM‐siRNA, n=6, and 101.1±1.0 to 109.8±0.5 mmHg in AT1‐siRNA, n=5). An immunohistochemical analysis of AT1‐siRNA‐injected rats showed a marked reduction in the expression of AT1 in the subfornical organ and paraventricular nucleus in comparison to SCM‐siRNA rats. These results suggest that the brain RAS is involved in the hypertensinogenic response produced by systemic ALDO administration. (Support: NIH HL–59676 and HL–62261)