bluuebunny/crossref_metadata_2025 huggingface dataset:
Aging and high intravascular pressure increase eNOS uncoupling
Authors: Yang Yang‐Ming , Huang An , Kaley Gabor , Sun Dong
Published: June 2021

eNOS uncoupling is an important mechanism that leads to endothelial dysfunction. Previously, we reported that shear stress‐induced release of NO in vessels of aged rats was significantly reduced, which was accompanied by increased production of superoxide. Furthermore, we found that in aged vessels superoxide production induced by high intravascular pressure was reduced by inhibition of NO synthase. In the present study, we investigated the influence of aging on eNOS uncoupling. Mesenteric arteries were isolated from young (3 month) and aged (24 month) C57 BL/6J mice. The expression of eNOS protein in young vs. aged mice was not significantly different. However, when we determined the ratio of eNOS monomers vs. dimers in young and aged mice, the aged mice had a significant increase in the monomer component. When isolated and perfused mesenteric arteries (∼200 μm) from aged mice were exposed to high intravascular pressure (180 mmHg) for 1 h, the ratio of eNOS monomers in these vessels was further elevated, and the elevation remained constant for 4 h. Interestingly, inhibition of NAD(P)H oxidase with apocynin (100 μM) protected the vessels from an increase in eNOS monomers. These data suggest that superoxide production may affect eNOS uncoupling, which could contribute to endothelial dysfunction in aged vessels. (Supported by NIH HL‐43023, HL‐68813 and HL‐070653)
Alternative Splicing of Dscam
Authors: Graveley Brenton R
Published: June 2021

Alternative splicing is an important means of regulating gene expression and increasing protein diversity. The arthropod Dscam gene is remarkably capable of generating tens of thousands if isoforms by the alternative splicing of nearly 100 exons. DSCAM plays an important role in both the wiring of the nervous system and in pathogen recognition in the immune system. Thus understanding the mechanisms by which Dscam splicing is regulated will lead to important insight into neurobiology and immunology. I will discuss our recent work aimed at understanding the mechanisms by which the inclusion of specific exons is regulated, the mechanisms of mutually exclusive splicing, and how this remarkable gene has evolved.
Evidence for crossreactivity of JAM‐C antibodies: Implications for cellular localization studies
Authors: Betanzos Abigail , Liang Tony A. , Severson Erick A. , Parkos Charles A.
Published: June 2021

Junctional Adhesion Molecule C (JAM‐C) is implicated in regulation of leukocyte migration, but detailed cellular localization studies are incomplete. Here, we used monoclonal (LUCA14, MAB1189 and Gi11) and polyclonal (AF11889 and 40–9000) antibodies to evaluate JAM‐C expression in some epithelial cell lines. By immunofluorescence (IF), all antibodies showed intercellular junctional staining and western blot (WB) analyses revealed a prominent band at 52kD which is larger than expected for JAM‐C (37kD). Interestingly, RT‐PCR revealed no JAM‐C transcripts in SK‐CO15 (SK), T84, HeLa and HPEF‐II cells, whereas abundant mRNA in platelets, Caco‐2, ARPE and JAM‐C transfected SK (JC‐SK) cells was detected. Using another recently described monoclonal antibody termed PACA4 (Raven biotech), WB of platelets, ARPE and JC‐SK cells revealed a single 37kD band, and it did not label epithelial cells that lack JAM‐C mRNA. Analyses by mass spectrometry identified the 52kD band as cytokeratin 8 (CK8). Further, siRNA‐mediated downregulation of CK8 in JAM‐C mRNA‐negative cells resulted in diminished junctional staining along with a reduction in the 52kD band. These results demonstrate that several anti‐JAM‐C antibodies crossreact with CK8 and suggest that cellular localization studies with such reagents should be interpreted with caution. Our results also suggest that mAb PACA4 is monospecific for JAM‐C.
Vascular endothelial growth factor (VEGF) receptors expresion and VEGF–mediated permeability of human amnion to calcium in chorioamnionitis (ChA)
Authors: Szukiewicz Dariusz , Maslinska Danuta , Stangret Aleksandra , Klimkiewicz Jakub , Pyzlak Michal
Published: June 2021

VEGF is a mediator of inflammation and an angiogenic factor. VEGF receptor type 1 (VEGFR1) regulates vascular permeability, while VEGFR2 is necessary for proliferation during the process of angiogenesis. ChA may change neovasculatization and permeability of placental membranes. We examined expression of VEGFR1 and VEGFR2 within amniotic epithelial cells and VEGF–mediated permeability of human amnion to calcium ions in ChA. 16 pieces of isolated extraplacental amnion obtained after near to term pregnancies complicated by ChA were compared with 16 gestationally matched amnion samples from normal pregnancies (Group I and II, respectively). Expression of VEGFR1 and VEGFR2 within amniotic epithelium was examined in calibrated areas of immunostained paraffin sections using morphometry for quantitative analysis. Diffusion of Ca++ was measured using a system of plastic container divided into two chambers by the mounted amnion. The effect of VEGF administration (doses range: 100–500 pg/ml) on the transport coefficient for Ca++ was examined in both groups. Expression of VEGFR1 was significantly increased in Group I and was positively correlated with increased VEGF‐mediated amnion permeability for Ca++ (p < 0.05). Our results indicate important influence of proinflammatory cytokine VEGF on calcium‐dependent biological phenomena, including preterm myometrial contractions

Supported by AM grant: 2M2‐W2‐07.
ACTIVATION OF CYTOSOLIC PHOSPHOLIPASE A2[alpha] THROUGH NITRIC OXIDE‐INDUCED S‐NITROSYLATION: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND CYCLOOXYGENASE‐2
Authors: Xu Lihong , Han Chang , Lim Kyu , Wu Tong
Published: June 2021

Cytosolic phospholipase A2α (cPLA2α) is the rate‐limiting key enzyme that releases arachidonic acid (AA) for eicosanoid production. This study describes that cPLA2α protein is S‐nitrosylated and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric oxide synthase (iNOS) in human epithelial cells induced cPLA2α S‐nitrosylation, enhanced its catalytic activity and increased the release of AA. The iNOS‐induced cPLA2α activation is blocked by the specific iNOS inhibitor, 1400W. Addition of the NO donor, GSNO, to isolated cell lysates induced S‐nitrosylation of cPLA2α, in vitro. Incubation of cultured cells with the iNOS substrate L‐Arginine and NO donor significantly increased cPLA2α activity and AA release. Furthermore, COX‐2 induction or expression markedly enhanced iNOS‐induced cPLA2α S‐nitrosylation and activation, leading to 9, 23, 20 fold increase of AA release and 100, 38, 88 fold of PGE2 production in A549, SG231, and HEK293 cells, respectively, whereas COX‐2 alone leads to less than 2 fold change. These results indicate that COX‐2 has the ability to enhance iNOS‐induced cPLA2α S‐nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA2α and COX‐2. These findings disclose a novel link among cPLA2α, iNOS and COX‐2 which form a multiprotein complex leading to cPLA2α S‐nitrosylation and activation.
ATPγS stimulates COX‐2 expression via p38 and p42/p44 MAPK in A549 cells
Authors: Wu Wan‐Ling , Yang Chuen‐Mao
Published: June 2021

In respiratory system, pulmonary alveolar epithelial cells play an important role in the first‐line defense of exteriors and contribute to inflammation. ATP is released from epithelial cells under physiological and pathological conditions. ATP has been shown to induce the expression of COX‐2 in airway epithelial cells and contributes to inflammatory responses. However, the mechanisms underlying ATP‐induced COX‐2 expression and PGE2 synthesis in human pulmonary A549 cells were not completely understood. In this study, we demonstrated that ATPγS induced COX‐2 protein and mRNA expression in a time‐ and concentration‐dependent manner, which was attenuated by pharmacological inhibitors of MEK1/2 (PD98059), p38 MAPK (SB202190), and NF‐κB (Bay11‐7082), revealed by RT‐PCR and Western blot analyses. Transfection with siRNAs of MEK1, p42, and p38 also inhibited ATPγS‐induced COX‐2 expression. In addition, ATPγS stimulated phosphorylation of p42/p44 MAPK and p38 in a time‐dependent manner, which was attenuated by pretreatment with PD98059 and SB202190. ATPγS‐induced NF‐κB translocation and IκBα degradation was attenuated by PD98059 and SB202190. These results demonstrated that ATPγS‐induced COX‐2 expression via activation of p42/p44 MAPK, p38 and NF‐κB in A549 cells.
Bowel Mucosa Lymphoid Tissue in Experimental DMH Administration
Authors: Cabrita Antonio Silverio , Farinha Rodrigo , Rafael Ana Isabel , Matos‐Beja Manuel Correia
Published: June 2021

The colon lamina propria is rich in lymphatic noduli (LN), which can occasionally become more proeminent in the mucosa. This can be caused by numerous stimuli. This work reports the changes found in the lymphoid tissue nodulae at the bowel mucosa level.

We used 60 Wistar male rats randomly distributed in three groups of twenty animals each: group I, kept with no manipulation; group II submitted to the administration of 15mg DMH/Kg twice a week during 8 weeks, after which were sacrificed; group III submitted to the administration of the same toxic and concentration, for 8 weeks, sacrificed 4 weeks after the last administration. The intestine was collected for histology. By morphometry we evaluated the percentage of each lymphatic nodule that is present in the mucosa area.

In the control group we didn't find LN occuping the mucosa area. In group II the LN area, for a 95% confidence interval varies between 211.1 and 234.7, and the percentage of LN area in the mucosa varies between 28.9% and 30.2%. The presence of LNs in the mucosa was 15% of the animals. In group III the LN area, for a 95% confidence interval varies between 236.1 and 290.1, and the percentage of LN area in the mucosa varies between varies between 24.7% and 28.8%. The presence of LNs in the mucosa was 35% of the animals.

According to these findings, this carcinogen increased the bowel lymphoid tissue present in the mucosa.
Suppressor of cytokine signaling (SOCS)‐1 but not SOCS‐3 inhibits MCP‐1 production in mouse lung epithelial cells
Authors: Chung Chun‐Shiang , Chen Yaping , Ayala Alfred
Published: June 2021

Cytokines play pivotal roles not only in normal cellular functions, but also in the pathogenesis of many diseases, such as acute lung injury (ALI). Epithelia cells in the lung are important for controlling the production of inflammatory mediators, which are critical to the pathogenesis of ALI. Understanding of positive/negative regulation of cytokine signaling may provide new therapeutic targets. SOCS proteins have been identified as feedback inhibitors of cytokine‐induced JAK/STAT signaling. However, the role of SOCS‐1/‐3 proteins in the regulation of cytokine production in lung epithelial cells is unclear. In this study, using lung epithelial cell lines we test whether SOCS‐1 or ‐3 contributes to the inhibition of cytokine production. Our initial results show that IFNγ, TNFα and LPS (to a less extent) induced SOCS‐1/‐3 gene and protein expression as well as production of IL‐6 and MCP‐1. To test whether SOCS proteins inhibit IL‐6 or MCP‐1 production, cells were transfected with siRNA of SOCS‐1 or −3, stimulated with IFNγ or TNFα, and IL‐6/MCP‐1release was measured. The data shows that silencing of SOCS‐1 expression increased MCP‐1 production as expected. However, SOCS‐3 gene knock down had no effects on MCP‐1/IL‐6 production. Together, these results indicate that SOCS‐1 and ‐3 differentially regulate MCP‐1/IL‐6 production in lung epithelial cells. (Shock‐Novo Nordisk Fellowship)
Lysosomal Dysfunction Promotes Autophagic Stress and NPC Death
Authors: Walls K.C , Zhang Jianhua , Geng Ying , Roth Kevin A
Published: June 2021

Autophagic pathways have been shown to regulate neural precursor cell (NPC) survival during nervous system development. To determine whether lysosomal dysfunction leads to increased autophagic vacuoles (AVs) and NPC death, we treated mouse cerebellar neural stem cells (C17.2 cell line) and FGF2‐expanded primary cerebellar NPCs with lysosomal dysfunction inducing agents chloroquine (CQ), bafilomycin A1 (Baf A1), and ammonium chloride (NH4Cl). CQ, NH4Cl, and Baf A1 treated NPCs underwent apoptosis, as evidenced by cytological features and cleaved caspase‐3 immunoreactivity. In comparison to wild‐type NPCs, Bax‐deficient NPCs exhibited significantly less caspase‐3 activation and cell death in response to lysosomal dysfunction inducing agents. These agents also produced increased levels of autophagy‐associated proteins LC3‐II and Beclin1. Cycloheximide, a protein synthesis inhibitor, attenuated AV formation in response to CQ as evidenced by decreased LC3‐II. However, cycloheximide had no effect on caspase‐3 activation or cell death indicating that AV accumulation is not necessary for CQ‐induced NPC death. p53‐deficient NPCs showed significant protection from CQ‐induced death compared to wild‐type NPCs. In total, our data demonstrate that lysosomal dysfunction leads to AV accumulation as well as p53‐ and Bax‐ dependent NPC death.
Effects of Poloxamer 188 (P‐188) on antioxidant status in rat skeletal muscle subjected to tourniquet (TK)‐induced ischemia/reperfusion (I/R) injury
Authors: Dubick Michael A , Barr Johnny L , Christy Robert J , Roe Janet L , Walters Thomas J
Published: June 2021

In our aim to reduce TK‐induced muscle edema and I/R injury, the present study investigated the effects of P‐188, a multi‐block copolymer surfactant reported to seal damaged membranes in a number of injury states, on antioxidant status in skeletal muscle subjected to I/R. Anesthetized rats (n=8–9/group) were subjected to 3 hr of TK‐induced ischemia. P‐188 (150 mg/kg) or vehicle was infused iv at 5 min before and at 240 min after reperfusion. Rats were then euthanized 16 hr later and lateral gastrocnemius muscle from the TK and contralateral (control) leg was collected for analysis. TK‐induced I/R resulted in nearly a 3‐fold increase in thiobarbituric acid reactive substances (TBARS) and a 34% increase in total antioxidant potential compared to control muscle. In addition, muscle glutathione (GSH) and GSH‐reductase activity were 53% and 24% lower in TK than control muscle, respectively. P‐188 treatment has no discernable effect on muscle antioxidant potential, TBARS or antioxidant enzymes in control muscle. In contrast, in TK muscle, P‐188 reduced the marked increase in TBARS by 23% and improved GSH concentrations, but did not significantly improve antioxidant enzyme activities. These data suggest that at the doses employed, P‐188 had modest antioxidant effects in agreement with about a 10% reduction in TK‐induced muscle edema and no effect on TK‐induced loss of muscle viability.
Myeloperoxidase suppresses apoptosis of human neutrophils
Authors: Kebir Driss El , Jozsef Levente , Pan Wanling , Filep Janos G
Published: June 2021

Myeloperoxidase (MPO), a heme protein abundantly expressed in the azurophilic granules in neutrophils mediates killing of bacteria and oxidative tissue injury. Since elevated plasma MPO levels and prolonged survival of neutrophils are characteristic features of inflammatory diseases, we studied the impact of MPO on neutrophil apoptosis. Culture of neutrophils with MPO at clinically relevant concentrations (10–160 nM) markedly enhanced neutrophil viability by suppressing apoptosis. These actions were prevented by an anti‐CD11b antibody, but not by 4‐aminobenzoic acid hydrazide, an inhibitor of the enzymatic activity of MPO. MPO evoked concurrent activation of the ERK and phosphatidylinositol 3‐kinase/Akt signaling pathways, leading to phosphorylation of Bad at Ser112 and Ser136, respectively. These led to prevention of collapse of mitochondrial transmembrane potential and cytochrome c release, resulting in decreased caspase‐3 activity. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3‐kinase almost completely blocked the responses to MPO. MPO and the pan‐caspase inhibitor z‐VAD‐fmk (20 μM) did not produce additive suppression of neutrophil apoptosis. Our results identify MPO as a survival signal for neutrophils by delaying intrinsic neutrophil apoptosis and thus may contribute to amplification of the inflammatory response. (Supported by CIHR MOP‐64283).
A prothrombogenic phenotype is exhibited in sickle cell transgenic mice
Authors: Gavins Felicity Nicola , Granger Neil D. , Buckingham Julia C.
Published: June 2021

Sickle cell disease (SCD) is a chronic genetic and lifelong disease, affecting the vasculature of different organs (1). This study, investigated the effects of SCD on thrombus formation.

Intravital microscopy was performed in the cerebral microcirculation of wild‐type (WT), sickle cell transgenic (SCD; ƒÒs) and WT/ƒÒs chimeric (positive control) mice (as previously described: 1). Thrombus formation was formed using the light/dye model in arterioles and venules. Flow cessation time was recorded.

Thrombus formation was reduced by 50% in SCD mice vs. WT in venules and arterioles. However, no difference was seen in WT/ƒÒs chimeric mice, suggesting the SCD mice response was not due to how they were generated.

The clinical implication of anti‐coagulant therapy in SCD mice was investigated. Both Hirudin and anti‐thrombin III were administered. Although Hirudin increased the time required for thrombus formation, the anti‐thrombin III inhibitor was more effective in SCD venules, suggesting thrombus formation is effected via thrombin, but is dependent upon the anti‐thrombin III pathway in venules.

These data further support the growing evidence for a pro‐inflammatory and pro‐thrombogenic microcirculation in SCD mice, which may lead to the identification of potential therapeutic targets in this painful and life‐threatening disease.

Supported by the BBSRC
Endothelial Expression of Beta‐1 Integrin Is Required for Embryonic Vascular Patterning and Postnatal Vascular Remodeling
Authors: lei li , Liu Dinggang , Huang Yan , Jovin Ion , Shai Shaw‐Yung , Kyriakides Themis , Ross Robert , Giordano Frank
Published: June 2021

The largest subgroup of integrins is that containing the β1 subunit. Global deletion of β1 integrin expression results in embryonic lethality at ~E5, a developmental time point too early to determine the effects of this integrin on vascular development. To elucidate the specific role of β1 integrin in the vasculature, we conditionally deleted the β1 gene in the endothelium using Cre/LoxP techniques. Homozygous deletion of β1 integrins in the endothelium resulted in failure of normal vascular patterning, severe fetal growth retardation, and embryonic death at E9.5‐10, although there were no overt effects on vasculogenesis (Fig. 1). Heterozygous endothelial β1 gene deletion did not diminish fetal or postnatal survival, but reduced β1 subunit expression in endothelial cells from adult mice approximately 40%. These mice demonstrated abnormal vascular remodeling in response to experimentally altered in vivo blood flow, and diminished vascularization in healing wounds (Fig. 2). These data demonstrate that endothelial expression of β1 integrin is required for developmental vascular patterning, and that endothelial β1 gene dosing has significant functional effects on vascular remodeling in the adult. Understanding how β1 integrin expression is modulated may have significant clinical importance.
Control of integrin‐mediated platelet adhesion by rigidity sensing at the surface of fibrin(ogen) matrix
Authors: Podolnikova Nataly P. , Bowen Benjamin , Lishko Valeriy , Podolnikov Andriy , Ugarova Tatiana
Published: June 2021

Recent evidence suggests that substrate rigidity controls cell adhesion through integrin‐mediated signaling. We have found that binding of soluble fibrinogen to the surface of insoluble fibrin matrix inhibits integrin‐mediated cell adhesion by creating an anti‐adhesive fibrinogen layer. To gain understanding of the mechanism underlying the differential ability of cells to adhere to the naked fibrin gel (rigid substrate) and to the loose fibrinogen layer formed on the surface of fibrin (soft substrate), we examined integrin αIIbβ3‐mediated signaling in platelets. Platelet adhesion to fibrin clots induced strong phosphorylation of FAK and Syk kinases as compared to adhesion on soft substrates. Also, the rigid, but not soft substrate induced recruitment of signaling molecules talin and skelemin to integrin‐containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. Differential platelet signaling was also observed when fibrinogen was adsorbed on plastic at low and high densities, a condition which mimics rigid and soft substrates, respectively. These data suggest that soluble fibrinogen prevents stable platelet adhesion to the fibrin clot by modifying its mechanical properties which results in insufficient signaling and reduced force generation.

NIH HL 63199
Gender differences in the handling of biologically‐active magnesium (Mg2+) and calcium (Ca2+) in short‐term (S‐T) dietary magnesium deficiency (MD); Implications for cardiovascular diseases (CVD)
Authors: Altura Bella T. , Shah Nilank C. , Liu Jian‐ping , Sica Anthony L. , Altura Burton M.
Published: June 2021

There has been a steady decline in the dietary intake of Mg in the USA. Is there a gender difference in the handling of Mg2+? To test this hypothesis, we induced S‐T MD (10% normal intake)in young ,mature male and female Wistar rats and used ISEs to measure, precisely, serum levels of Mg2+, Ca2+, Ca2+/Mg2+ ratios and total Mg (tMg).The MD‐animals were allowed to drink triply‐distilled water containing one of five different levels of Mg ,while control animals received a normal Mg‐containing diet The data indicate that male and female rats, on normal Mg diets ,exhibit identical serum levels of Mg2+(0.64 +/− 0.02 vs.0.63 +/− 0.3 mM),Ca2+(1.47 +/− 0.08 vs 1.43 +/− 0.03 mM),and Ca2+/Mg2+ ratios (2.30 vs.2.17). The MD‐male and female rats exhibited different Mg2+(0.26+/−0.04 vs. 0.36+/−0.06 mM),tMg and Ca2+/Mg2+ ratios ( 6.19 vs 4.3) from one another and the controls ( p< 0.01). Feeding water‐borne Mg failed to restore Mg2+ or tMg to normal serum levels in male animals ,but the higher water‐borne levels of Mg in females were able to restore serum Mg2+ and tMg to normal.We demonstrate ,for the first time, that ;1) mature male MD‐ rats do not have an ability to mobilize biologically‐active Mg2+ as do female mature MD‐ rats; 2) male rats exhibit high serum Ca2+/Mg2+ ratios in a S‐T MD state, which would result in increased risk for the development of CVD.
Identification of premature ovarian failure‐associated genes using linkage disequilibrium‐based genome‐wide association study
Authors: Kang HyunJun , Lee Seung Ku , Kim Hyoun Geun , Kim Kyoung‐Yeon , Kim Jeong‐Geun , Oh Ah Reum , Pyun Jung A , Kim Min‐Ho , Won SeongSik , Kang Eun Joo , Lee Sook‐Hwan , Kwack KyuBum
Published: June 2021

Premature ovarian failure (POF) as known to be a heterogeneous and polygenic disorder is the cessation of menstrual cycle before the age of 40. It was characterized by primary amenorrhea (absent menarche) and secondary amenorrhea (lack of gonadotrophin stimulation). Even though there were many of reports, physiological mechanism and genes contributed to POF still are in mist. To identify genes considered to be associated with POF, genome‐wide association study (GWAS) based on linkage disequilibrium (LD) mapping was performed with 24 patients with POF and their 24 matched controls. In the present study, about 100 genes were identified to be associated with POF. Among them, two genes revealed the best strong association with POF, which were laminin, gamma 1 (LAMC1) and branched chain alpha‐keto acid dehydrogenase, E1‐beta subunit (BCKDHB) genes. Through the LD analysis, complete LD (|D’|=1,γ2 ≠ 1) block was formed throughout LAMC1 gene, and the same block was formed focused on exon 10 including alternative splicing site in BCKDHB gene. Haplotypes reconstructed from each blocks showed significant association with POF after statistical correction by multiple testing. The results in our study suggest that these genes may contribute to increase the risk for POF. With regard to the concepts of LD, it is suggested that causal variants may exist in POF‐susceptible haplotypes. We hope that our findings propose a new direction in this field.
A data mining analysis of pathological findings in 11259 placentas
Authors: Zuo Zhuang , Kahn Andrea G. , Tucker J. Allan
Published: June 2021

To systematically review the pathological findings in placentas, we identified a data set of 11259 cases from 1996 to 2007 in our database. The diagnoses, along with available clinical information, were extracted from the reports by text mining, and parsed into a relational database. The associations among various epidemiologic and pathologic features with clinical outcomes were examined by data mining analysis. The maternal age at delivery ranged from 12 to 49 years (mean 24). Eight diagnostic categories included 26 items, such as no significant pathological change (55%), infarct (16%), villitis (15%), and accelerated maturation (9%). Statistically significant findings (p<0.0001) included: increased maternal age since 2001; increased percentage of cases with pathological findings in women ≥30 years; prematurity associated with age > 26 years, obliterative fetal vasculopathy, inflammation, infarct, thrombosis, marginal umbilical cord insertion, and previous preterm birth; accelerated maturation associated with inflammation, obliterative fetal vasculopathy, marginal umbilical cord insertion, but negatively correlated with calcification. Plotted by maternal age versus gestational age, inflammatory processes and circulatory problems clustered in distinct areas. In conclusion, this study showed that data mining is a valuable approach to discover hidden patterns in large clinical data sets.
Antibody identification by Luminex technology in renal allotransplant recipients
Authors: Lewis Robert , Cruse Julius , Sanders Catherine , Webb Rachel , Suggs Jeanann , Thomson Wendy
Published: June 2021

The alloimmune response induced by HLA histoincompatibility between donor and recipient is a primary source of rejection in renal allotransplant recipients. Newer methods of detecting transplant recipient antibodies include Luminex and flow cytometric technologies. In the present investigation, 45 renal allotransplant HLA antibody screens were performed using both flow cytometric screen beads and Luminex screen beads, and the results from both methods were compared. The Luminex detected antibodies that the flow cytometric beads failed to detect in 15 cases. Of these cases, 5 recipients had class I antibodies, 5 had class II antibodies, and 5 others had both class I and class II antibodies. Of the class I antibodies detected, 7 were A1 CREG antibodies, 1 was an A2 antibody, 4 were A10 CREG antibodies, 1 was an A19 CREG antibody, 5 were B5 CREG antibodies, 3 were B7 CREG antibodies, 2 were B8 CREG antibodies, and 2 were B12 CREG antibodies. Donor‐specific antibodies were present in 7 of the 15 cases. The flow cytometric beads detected an antibody in 1 case that the Luminex did not which after analysis was determined to be a false positive. Our results suggest that Luminex antibody screen beads may be better detectors of certain CREG group antibodies. These data indicate that using Luminex beads rather than flow cytometric screen beads may increase the sensitivity of antibody detection that could lead to allograft rejection.
Analyses of Jagged1 and Notch1 in Rat‐ and Human‐ Hepatocyte Culture: Differentiation and Proliferation Conditions affect receptor and ligand of Notch Pathway.
Authors: Koehler Christoph , Bell Aaron W. , Bowen William C. , Michalopoulos George K.
Published: June 2021

The Notch‐signaling pathway controls cell fate determination and differentiation, and its dysfunction is associated with Alagille syndrome or hepatocellular carcinoma. Our previous studies have shown that Notch and Jagged‐ siRNA treated rat liver reduces the ability in proliferation and regeneration following partial hepatectomy. To determine the putative role of Notch and Jagged in proliferation of hepatocytes, we analyzed expression of Jagged1 and Notch1 in the primary cultures of rat‐ and human hepatocytes. The hepatocytes were cultured in the presence or absence of HGF, EGF, TGFalpha, VEGF, Dexamethasone or Matrigel. Real‐time PCR and Western Blot analyses were performed to detect levels of Notch‐1, Jagged‐1 and target gene Hes‐1. Our results show that proliferative conditions for hepatocytes increase expression of Notch, Jagged or Hes. Whereas glucocorticoid induce expression of Notch and proliferation of endothelial cells, hepatocytes differentiate due to glucocorticoid induced downregulation of Notch. Hepatocytes surrounded by extracellular matrix downregulate Notch‐signaling and promote differentiation in vitro. The above results indicate that Notch signaling depend on factors, which are involved in differentiation or proliferation of hepatocytes. This supports our hypothesis that an equilibrium of cytokines and Notch‐signaling are necessary in controlled liver regeneration.
PTX‐insensitive Gz transduction pathway contributes to buprenorphine‐induced supraspinal, but not spinal, antinociception
Authors: DING ZHE , RAFFA ROBERT B
Published: June 2021

Buprenorphine is known as an opioid analgesic. Although it shares some of the general characteristics of other opioids, it displays preclinical pharmacology and clinical attributes distinct from opioids such as morphine and fentanyl. As a way of elucidating some of the differences, we investigated the contribution of G proteins in buprenorphine‐induced antinociception. Subcutaneous administration of buprenorphine, morphine or fentanyl induced antinociception in the warm‐water (48¡ãC) tail‐dip/flick test in mice in a dose‐related manner. The antinociception induced by all three agonists was attenuated by intrathecal (i.t.) administration of either pertussis toxin (PTX) (1 mg, 48h) or the opioid receptor antagonist naloxone (20 mg). However, intracerebroventricular (i.c.v.) PTX or naloxone administered attenuated only morphine‐ and fentanyl‐induced antinociception. Pretreatment with Gz antisense (14.6 mg, 24 h) i.t. had no effect on any agonist‐induced antinociception. However, pretreatment with Gz antisense (but not control) i.c.v. inhibited buprenorphine‐induced, but not morphine‐ or fentanyl‐induced antinociception. These results reveal that a PTX‐insensitive, Gz transduction pathway plays an important role in buprenorphine‐induced supraspinal antinociception distinct from, or to a greater degree than, morphine or fentanyl.

This work was supported by Grünenthal GmbH (Germany).
The 5‐HT7Dro Serotonin Receptor: Expression in the CNS and Function
Authors: Becnel Jaime T , Johnson Oralee , Nichols Charles D.
Published: June 2021

Serotonin (5‐HT) influences behaviors that include circadian, sleep, appetite, aggression, locomotion, perception, and sexual behavior. The effects of 5‐HT are mediated through G‐protein coupled receptors, which initiate multiple effector pathways. Disregulation of 5‐HT signaling in humans has been implicated in neuropsychiatric disorders including depression, anxiety disorders, anorexia and schizophrenia. The fruit fly, Drosophila melanogaster, expresses orthologs of three mammalian 5‐HT receptor subtypes: 5‐HT1A, 5‐HT2, and 5‐HT7. The 5‐HT7Dro receptor is orthologous to the mammalian 5‐HT7 receptor and activates adenylate cyclase through Gαs. We show that in fly 3rd instar larvae brain, 5‐HT7Dro expression is observed in discreet populations of neurons in the hemispheres as well as in neurons of the ventral ganglia. In adult brains, 5‐HT7Dro is highly expressed in neurons that innervate the ellipsoid body, a region believed to be involved in higher order behaviors. 5‐HT7Dro neurons also cluster with PDF‐positive circadian pacemaker neurons. Pharmacological studies using the 5‐HT1A/7 receptor agonist 8‐OH‐DPAT with the 5‐HT1A receptor antagonist WAY100635 eliminated circadian activity. These results suggest that 5‐HT7Dro may modulate circadian activity in the fly. Further studies to determine the role of 5‐HT7Dro in additional behaviors are ongoing. Supported by NIMH 1R21MH078454
Understanding receptor selectivity and ligand stereochemistry of insecticides and therapeutic agents for nicotinic acetylcholine receptor subtypes
Authors: Yamauchi John , Talley Todd , Park John , Tomizawa Motohiro , Casida John , Harel Michal , Taylor Palmer
Published: June 2021

Neonicotinoids are insecticides selective for nicotinic acetylcholine receptors (nAChRs) in agricultural pests having a far lower affinity for vertebrate receptors. Additional understanding for the basis of receptor selectivity with the nAChRs is crucial for ascertaining whether insecticides are responsible for the bee “colony collapse disorder” or other toxicities in insect populations. Moreover receptor selectivity is integral to improved efficacy of new nicotinic therapeutic agents. The acetylcholine binding protein (AChBP), through mutagenesis modifications, can serve as a template for design of new therapeutic agents and insecticides. Its chief advantage stems from its solubility, making complexes amenable to crystallography and physical studies. Ligand dissociation constants were determined for steriosomers with radio‐ligand binding assays, using [3H]‐Epibatidine competing for purified Aplysia or Lymnaea AChBP sites. We have obtained evidence that stereochemistry in alkaloid, nicotinoid, and neonicotinoid ligands results in substantial differences of binding affinity to specific AChBPs. These data will be compared with crystallographic data to ascertain determinants essential for agonist and antagonist selectivity.

Support from (R37‐GM18360, F32 NS043063, R01 ES08424, GM07752‐29)
Soluble templates for drug design: the acetylcholine binding proteins and freeze‐frame, “click” chemistry for in situ synthesis of nicotinic ligands
Authors: Talley Todd T , Fosting Joseph R , Weide Timo , Grimster Neil , Ho Kwok‐Yiu , Fokin Valery V , Taylor Palmer W
Published: June 2021

Our ongoing crystallographic and structure‐activity studies of the acetylcholine binding proteins provide insights into the ligand binding domain of nicotinic receptors as well as other members of the Cys‐loop family of receptors. Our current study combines traditional structure guided ligand design with in situ freeze frame, “click” chemistry techniques to construct a library of novel ligands. This strategy involves the design and synthesis of building block ligands of moderate to high affinity that contain either acetylene or azide functionalities. These functional groups are essentially unreactive under normal aqueous conditions. However, when they are brought into proximity of one another within the binding site of a macromolecule, they undergo a biorthogonal cycloaddition joining the building blocks through a triazole ring of specific regiochemistry. Hence the target protein is directly involved in the synthesis of multisubstrate adducts tailored to the specific ligand binding environment. Thus far, we have generated a number of high affinity ligands (Kd < 1nM) that demonstrate significant specificity. Coupled with crystallographic data, these compounds provide leads for the development of a structural base for novel nicotinic agonists and antagonists.
Expression of Modd muscarinic acetylcholine receptors in the retinas of bluegill and zebra fish
Authors: Crittenden Elizabeth Lyla , Capalbo Elizabeth L , García Dana M
Published: June 2021

Light adaptation in most vertebrates occurs through adjustments in pupil diameter. Teleosts, however, have a fixed pupil size, so they rely on retinomotor movements to regulate the amount of light incident on the photoreceptors. Retinomotor movements include photoreceptor contraction and elongation as well as the aggregation and dispersion of membrane bound pigment granules located in the retinal pigment epithelium. Previous work has shown that pigment granule dispersion can be induced using the neurotransmitter acetylcholine acting via muscarinic receptors and that dispersion can be inhibited by Modd receptor specific antagonists. In this study we demonstrate Modd receptor expression using immunolabeling with an anti M3 antibody and with in situ hybridization using probes that target the M5 receptor. Labeling with the M3 antibody was seen multiple layers of the bluegill retina and was supported by Western blot analysis using bluegill and zebrafish tissue. In situ hybridization of the M5 sequence indicated a similar distribution of the receptor as that seen with the antibody. These results suggest that multiple muscarinic receptor subtypes are expressed in the fish retina, and lay the foundation for future studies to elucidate the role of muscarinic signaling pathways in the retina, especially as they pertain to light adaptation.
Inhibitory activity of Thuja orientalis on TNF‐α‐induced vascular cell adhesion in HUVECs
Authors: Moon Mi Kyoung , Kang Dae Gill , Lee Yun Jung , Kim Jin Sook , Lee Ho Sub
Published: June 2021

Vascular inflammation is involved in the initiation and progression of cardiovascular disease including atherosclerosis. In this study, the anti‐vascular inflammatory activity of an aqueous extract of Thuja orientalis (ATO) and its possible mechanisms were investigated in human umbilical vein endothelial cells (HUVECs). Preincubation of HUVECs with ATO (20 μg/ml) suppressed the protein and mRNA expression levels of adhesion molecules including intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), E‐selectin. Preincubation of ATO inhibited tumor necrosis factor‐alpha (TNF‐α)‐induced nuclear factor‐kappaB (NF‐κB) p65 translocation into the nucleus and the phosphorylation of inhibitory factor κB (IκB‐α). ATO also inhibited U937 monocyte adhesion to HUVECs stimulated by TNF‐α, suggesting that it may inhibit the binding of monocyte to endothelium. Furthermore, ATO significantly inhibited TNF‐α‐induced production of intracellular reactive oxygen species (ROS). Overall, ATO has an anti‐inflammatory activity, which is, at least in part, the result of it reducing the TNF‐α‐induced endothelial adhesion to monocyte by inhibiting intracellular ROS production, NF‐κB activation, and cell adhesion molecule in HUVECs.
Benzo[a]pyrene sensitizes mouse aortic endothelial cells to oxidized lipids
Authors: Jules George , Zhou LiChun , Yang Hong , Guo ZhongMao
Published: June 2021

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant arising from the incomplete combustion of organic substances, and is known to cause many diseases including atherosclerosis. It has also been shown that BaP activates phospholipase A2, an enzyme necessary for the synthesis of lysophosphatidylcholine (LPC), an oxidized lipid known to promote atherosclerosis. Several studies have shown that both LPC and BaP promote endothelial cell (EC) death through the production of reactive oxygen species such as hydrogen peroxide, a relatively stable molecule. However, the mechanism by which both LPC and BaP promote EC death is not well defined. Preliminary data from our laboratory shows that BaP increases sensitivity of mouse aortic endothelial cell (MAECs) to LPC. Therefore the central hypothesis to be tested in this study is that BaP increases lysophosphatidylcholine (LPC) induced cell death in mouse aortic endothelial cells (MAECs) through the production of reactive oxygen species such as hydrogen peroxide.

To test this hypothesis, cell viability, membrane integrity, and apoptotic cell markers will be measured in MAECs obtained from wild‐type mice and mice overexpressing the human catalase gene (hCatTg+/0). Furthermore, phospholipase A2 enzyme activity will be examined in hCatTg+/0 and wild‐type ECs based on protein level, and the phosphorylation status of the protein. Since catalase is an antioxidant that converts H2O2 to water and oxygen, I expect to find cell death as well as phospholipase A2 activity to be decreased in hCatTg+/0 ECs compared to wild‐type cells.
Lercanidipine (LER) improves the endothelial dysfunction and prevents the vascular remodeling in diabetic rats
Authors: Martinez Márcio Luís Lombardi , Fernandes Karla , Bendhack Lusiane Maria , Tanus‐Santos José Eduardo
Published: June 2021

Objective: We investigated whether pretreatment with LER affects the vascular reactivity to Acethylcoline (Ach) and prevents the vascular remodeling in Diabetic rats.

Methods: Male Wistar rats (180–200 g) were used in this study. Insulin‐dependent diabetes was induced (alloxan 40 mg/kg), whereas in the control animals was injected vehicle only.

LER (2,5 mg/kg/day) was added daily to drinking water, starting 3 weeks after induction of diabetes and maintained for additional six weeks. The animals of this study were divided into four groups as follows: control (C); (b) treated control (CL); diabetic (D); treated diabetic (DL). Measures of arterial pressure (SBP – tail cuff method) and experiments of vascular reactivity, gelatin zymography and quantitative RT‐PCR were performed.

Results: SBP was increased in D group, and the treatment with LER attenuated the SBP in DL group. In the same way, LER restored the vascular reactivity to Ach in DL group. Moreover, increased MMP‐2 activity and expression was found in the aortas of D group, and LER reduced the MMP‐2 activity in DL group. In addition, an increase in the ratio of MMP‐2/TIMP‐2 mRNA expression was found in D group, whereas this ratio was reduced in DL group.

Conclusion: Our results show that LER produces antihypertensive effects and reverses, at least in part, the vascular changes associated with diabetes in rats.

Financial support: FAPESP‐CAPES‐CNPq
Monitoring development and prevention of nitrate tolerance (tachyphylaxis) in healthy volunteers with monocytic ALDH‐2 activity as a marker
Authors: Daiber Andreas , Wenzel Philip , Mäthner Falk , Göbel Sebastian , Warnholtz Ascan , Ostad Mir Abolfazl , Oelze Matthias , Stalleicken Dirk , Münzel Thomas
Published: June 2021

Recent experimental data suggest that cotreatment with reduced lipoic acid can ameliorate nitrate tolerance, a process that involves oxidative inhibition of the bioactivating enzyme, the mitochondrial aldehyde dehydrogenase (ALDH‐2). We sought to determine whether this applies to humans. 8 Healthy male volunteers were assigned to take a single dose of glycerol trinitrate (GTN, 0.8mg), pentaerithrityl tetranitrate (PETN, 80mg) or GTN plus pretreatment of alpha‐lipoic acid (LA, 600mg, 30min in advance). After maximum plasma‐concentrations were reached (30min for GTN and GTN‐LA, 120min for PETN), venous blood was sampled. We assessed the antioxidative capacity of serum by a fluorescence assay, monocyte ALDH‐2 activity and biotransformation of a trinitrate‐chromophore by HPLC methods. As compared to controls, GTN resulted in a 60% decrease in monocytic ALDH‐2 activity (that could be partially restored ex vivo by dithiothreitol) and 30% reduction of nitrate bioactivation as well as a 25% decrease in antioxidative capacity of serum. These effects were significantly improved by pretreatment with LA. Treatment with PETN did not impair ALDH‐2 activity, nitrate bioactivation or serum antioxidative capacity. We conclude that treatment with a single dose of GTN but not PETN acutely inhibits ALDH‐2 acitivity and nitrate bioactivation in human volunteers. The GTN effects could be ameliorated by LA pretreatment.
High fructose diet activates plasma ACE and renal ACE2
Authors: Senador Danielle , Key Mary , Irigoyen Maria Claudia , Elased Khalid M , Morris Mariana
Published: June 2021

Dietary fructose in mice increases blood pressure via activation of the renin angiotensin system. To extend these findings, we tested the effect of an angiotensin AT1 antagonist, losartan, on cardiovascular parameters and ACE/ACE2 activity. Male C57BL mice were fed a 60% fructose diet for 10 weeks. Telemetric probes were implanted at week 8 and losartan (30 mg/kg/day) was given in the drinking water during the last week. Desorption/Ionization Mass Spectrometry (SELDI‐TOF‐MS) and fluorigenic assays were used to evaluate plasma ACE and kidney ACE2 activity, respectively. The SELDI‐TOF‐MS proteolytic assays use endogenous Ang substrates; Ang I for ACE and Ang II for ACE2. Losartan reduced MAP by 16% (117±1 to 99±3 mmHg) in the fructose group, while it had no effect in controls. There were no alterations in HR. Plasma Ang II and ACE activity were higher in the fructose group (Ang II: 12±3 vs. 37±8 pg/ml; ACE: 1.12±0.64 vs. 4.67±1.3; AngII/Ang I). There was no alteration in kidney ACE activity between groups. Kidney ACE2 activity was increased in fructose group (2.83±0.231 vs. 3.94±0.119 pmol/hr/μg protein; control vs fructose). Fructose consumption enhanced the BP lowering effect of losartan. Increased renal ACE2 activity could play a renoprotective role and/or could be a feedback response to counteract the increased BP and plasma ACE activity observed in fructose fed mice.
Phosphorylation of carnitine palmitoyltransferase‐1 in heart mitochondria
Authors: Sharma Vijay , Parsons Hannah , Allard Michael F , McNeill John H
Published: June 2021

Aim: Carnitine palmitoyltransferase‐1 (CPT‐1) catalyzes the entry of long‐chain acyl CoA's to the cytoplasm, an important control step in fatty acid oxidation. Previous studies from our laboratory indicate that the maximum activity and malonyl CoA sensitivity of CPT‐1 are acutely regulated by metoprolol in the rat heart. The aim of the present study was to determine whether incubation of isolated mitochondria from left ventricular tissue with protein kinase A, Akt or calcium/ calmodulin‐dependent protein kinase alters the maximum activity or malonyl CoA sensitivity of CPT‐1. Methods: Isolated mitochondria from normal Wistar rats were incubated with purified preparations of PKA, CAMK II or Akt for 10 minutes. Phosphorylation of CPT‐1 isoforms was measured by co‐immunoprecipitation with phosphothreonine and phosphoserine antibodies, and the activity and malonyl CoA sensitivity of CPT‐1 was assayed. Results: PKA and CAMK‐II phosphorylated CPT‐1. PKA decreased CPT‐1 malonyl CoA sensitivity whereas CAMK‐II increased CPT‐1 malonyl CoA sensitivity. There was no effect on the maximum activity of CPT‐1. Conclusions: PKA and CAMK‐II regulate CPT‐1 sensitivity in isolated heart mitochondria in vitro. These kinases may be counter‐regulatory in the control of CPT‐1 malonyl CoA sensitivity.