A monoclonal antibody that recognizes Epstein-Barr virus nuclear antigen 2 (EBNA 2) amino acids 1–58 does not react with EBNA 2 in native form,consistent with the self-association of EBNA 2 through the amino-terminus

Summary. We have generated a mouse IgG₁ monoclonal antibody (mAb) that recognizes amino acids 1–58 of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA 2) of type 1 EBV strain B95-8. mAb Y101 also reacted with EBNA 2 of EBV type 2 strains MISP and Jijoye in immunoblots, whereas Jijoye EBNA 2 was not detected by the widely used mAb PE2. mAb Y101, in contrast to mAb PE2, reacted with faster migrated, hypophosphorylated proteins of type 1 EBNA 2 as intensely as slower migrated, hyperphosphorylated ones. mAb Y101 did not react in fixed-cell immunostaining or cell extract immunoprecipitation. The results implicate that the amino-terminal epitope is not exposed in a native form, consistent with the previously reported idea of self-association of EBNA 2 through the amino-terminus. mAb Y101 is the first mAb to the EBNA 2 amino-terminus and will be useful for further analyses of the structure and function of EBNA 2.     

BAMHI DNA Fragment H-Polymorphism of Epstein–Barr Virus is Associated with the Mutations Present in an 89 BP Sequence Localized in EBNA2 Gene

To characterize the genotypes of Epstein-Barr virus (EBV) isolate present in North Africa, viruses were isolated from B-lymphoblastoid cell lines established from the saliva of both Algerian Nasopharyngeal Carcinoma (NPC) patients and EBV-positive normal individuals, Algerian Burkitt's lymphoma cell lines, and NPC biopsies. By nucleotide sequence analysis, we showed that there were two specific missense mutations in an 89 bp region of EBNA2 gene at position 49390-49479 of the EBV genome: a mutation at 49449 (C-->A) and another mutation at 49444 (T-->C), changing their amino acid sequence. The first mutation was found in all B cell lines established from the saliva and 50% of BL cell lines, as well as the W91 cell line, while the second mutation was found in EBV isolates from NPC biopsies, BL cell lines and the M-ABA isolate. A PCR-RFLP analysis on the BamHI DNA fragment H showed that the Hl-H2-polymorphism was specifically associated with M-ABA-like mutation, while H-polymorphism was linked with W91-like mutation. The latter was not identified in NPC biopsies, but was found rather in saliva from NPC patients, normal individuals and BL cell lines. The M-ABA-like mutation, on the other hand, was found in 100% of NPC biopsies and some BL cell lines. This suggests that EBV with H1-H2-polymorphism is tightly implicated in NPC development in North Africa rather than EBV with H-polymorphism.     

EB病毒基因LMP1和EBNA2对支气管上皮细胞的转化

目的 观察Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）对支气管上皮细胞的转化作用。方法 用ＥＢＮＡ２和ＬＭＰＩ的真核表达质粒同时转染永生化人胚支气管上皮细胞系ＴＲ,经潮霉素Ｂ筛选得到稳定转化细胞ＴＲ／ＬＭＰＩ－ＥＢＮＡ２。结果 原位杂效和Ｗｅｓｔｅｍ ｂｌｏｔ证实ＴＲ／ＬＭＰＩ－ＥＢＮＡ２有ＥＢＮＡ２和ＬＭＰＩ的ｍＲＮＡ和蛋白表达。生长曲线,ＭＴＴ显示细胞增殖能力增强,软琼脂集落形成率ＴＲ／ＬＭＰＩ－Ｅ     

Role of the Pyk2–MAP Kinase–cPLA2 Signaling Pathway in Shear-Dependent Platelet Aggregation

Mechanisms of shear-induced platelet aggregation are not established. Data that ristocetin-induced von Willebrand factor (VWF) binding to glycoprotein (Gp) Ibalpha activates proline-rich tyrosine kinase 2 (Pyk2) and extracellular-regulated kinase (ERK) has led to speculation that these events are coupled and that a MAP kinase may activate cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release. To test this hypothesis and clarify the role of AA metabolism in shear-induced VWF-dependent platelet aggregation, we examined Pyk2, ERK1/2, and p38 phosphorylation, and arachidonic acid release and metabolism in platelets subjected to pathological shear stress in vitro. We observe tyrosine phosphorylation of Pyk2, p38, and ERK1/2 but no measurable increase in free AA, 12-hydroxyeicosatetraenoic acid, or thromboxane A2. Inhibitors of ERK, p38, or cyclooxygenase activation fail to affect shear-induced platelet aggregation. When washed platelets are aspirin-pretreated, arachidonic acid release becomes measurable and aggregation at 60 and 120 s is attenuated. These data indicate that shear-induced VWF binding to platelet GpIb-IX-V activates Pyk2, ERK1/2, p38, and cPLA2, but that the magnitude of these responses is below the threshold needed to enhance shear-induced VWF-dependent platelet aggregation in the presence of plasma. These results provide a mechanistic basis for the long-standing observation that shear-dependent platelet aggregation is unaffected by the antiplatelet drug aspirin.     

Genetic disruption of the oncogenic HMGA2–PLAG1–IGF2 pathway causes fetal growth restriction

PurposeFetal growth is a complex process involving maternal, placental and fetal factors. The etiology of fetal growth retardation remains unknown in many cases. The aim of this study is to identify novel human mutations and genes related to Silver–Russell syndrome (SRS), a syndromic form of fetal growth retardation, usually caused by epigenetic downregulation of the potent fetal growth factor IGF2.MethodsWhole-exome sequencing was carried out on members of an SRS familial case. The candidate gene from the familial case and two other genes were screened by targeted high-throughput sequencing in a large cohort of suspected SRS patients. Functional experiments were then used to link these genes into a regulatory pathway.ResultsWe report the first mutations of the PLAG1 gene in humans, as well as new mutations in HMGA2 and IGF2 in six sporadic and/or familial cases of SRS. We demonstrate that HMGA2 regulates IGF2 expression through PLAG1 and in a PLAG1-independent manner.ConclusionGenetic defects of the HMGA2–PLAG1–IGF2 pathway can lead to fetal and postnatal growth restriction, highlighting the role of this oncogenic pathway in the fine regulation of physiological fetal/postnatal growth. This work defines new genetic causes of SRS, important for genetic counseling.     

Inhibition of the autocrine IL-6–JAK2–STAT3–calprotectin axis as targeted therapy for HR?/HER2+ breast cancers

HER2-positive (HER2⁺) breast adenocarcinomas are a heterogeneous group in which hormone receptor (HR) status influences therapeutic decisions and patient outcome. By combining genome-wide RNAi screens with regulatory network analysis, we identified STAT3 as a critically activated master regulator of HR⁻/HER2⁺ tumors, eliciting tumor dependency in these cells. Mechanistically, HR⁻/HER2⁺ cells secrete high levels of the interleukin-6 (IL-6) cytokine, inducing the activation of STAT3, which in turn promotes a second autocrine stimulus to increase S100A8/9 complex (calprotectin) production and secretion. Increased calprotectin levels activate signaling pathways involved in proliferation and resistance. Importantly, we demonstrated that inhibition of the IL-6–Janus kinase 2 (JAK2)–STAT3–calprotectin axis with FDA-approved drugs, alone and in combination with HER2 inhibitors, reduced the tumorigenicity of HR⁻/HER2⁺ breast cancers, opening novel targeted therapeutic opportunities.     

Connection types between the spinal root of the accessory nerve and the posterior roots of the C2–C6 spinal nerves

Purpose  The aim of this study was to demonstrate the connection types and frequency between the accessory nerve and the posterior roots of the C2–C6 cervical nerves. Methods  The cranial cervical regions of 49 specimens from 27 human cadavers were used for the present study under an operating microscope. Results  Five different connection types between the accessory nerve and the posterior roots of the cervical nerves were recorded and photographed (types A–F). One of these types was not described previously in literature (type F). All connections between the posterior roots of the C2–C6 spinal nerves and the accessory nerve were at the level of the C2 segment. Type B was the most frequently seen type in our series. One of the rootlets of the cervical posterior root joined the accessory nerve without a connection to the spinal cord in type B. Conclusions  The clinical importance of these connections is especially noticed during the radical neck dissection as it may lead to the development of the shoulder-arm syndrome.     

Epstein－Barr病毒核抗原Ⅱ（EBNA2）基因免疫的初步研究

利用基因免疫技术，将重组质粒ＰＳＧ５－ＥＢＮＡ２注入Ｂａｌｂ／Ｃ小鼠肌肉中，于第２、４、８周检测鼠血清中抗－ＥＢ病毒核蛋白抗原Ⅱ的特异抗体，结果表明，８３％（５／６）的免疫小鼠产生特异抗体，且抗体滴度随时间变化增高。     

Eostein—Barr病毒核抗原Ⅱ（EBNA2）基因免疫的初…

利用基因免疫技术，将重组质粒ｐＳＧ５－ＥＢＮＡ２注入Ｂａｌｂ／Ｃ小鼠肌肉中，于第２、４、８周检测鼠血清中抗－ＥＢ病毒核蛋白抗原Ⅱ的特异抗体，结果表明，８３％的免疫小鼠产生特异抗体，且抗体滴度随时间变化增高。     

EBNA1在鼻咽癌发生中细胞免疫应答的研究

EB病毒核心抗原1（EB nuclear antigen1,EBNA1）是唯一一个在EB病毒（Epstein-Barr Virus,EBV）潜伏感染和活化状态中均表达的抗原,也是在鼻咽癌（Nasopharyngealcarcinoma,NPC）组织中表达的几个EB病毒抗原之一.早期研究显示EBNA1为DNA结合蛋白,在促进细胞转化、EBV基因组稳定、复制、基因的表达方面起着重要的作用,并可以诱导体液免疫应答.近来研究表明在EBNAI的C末端含有CD4＋T细胞表位,在诱导细胞免疫应答方面起着重要作用,鉴此,本文对EBNA1在NPC发生中细胞免疫应答方面的研究做一综述.     

Methamphetamine-associated dysregulation of the hypothalamic–pituitary–thyroid axis

Methamphetamine and HIV impair thyroid function, but few studies have investigated their combined effects on thyroid dysregulation. This study examined the associations of methamphetamine use alone and in combination with HIV on thyroid function among men in South Florida. Measures of thyroid function in methamphetamine-using, HIV-infected (METH+HIV+; n?=?127) and HIV-negative (METH+HIV?; n?=?46) men who have sex with men (MSM) were compared to non-methamphetamine-using, HIV-negative men (METH?HIV?; n?=?136). Thyroid function was dysregulated in methamphetamine-using MSM, irrespective of HIV status. Both meth-using groups had greater odds of abnormal thyroid stimulating hormone levels and significantly higher mean free triiodothyronine (T3) levels. Elevated free T3 was associated with greater depressive symptoms. Overall, outcomes have important implications for assessment of thyroid function in methamphetamine users, particularly among those presenting with depression.     

ACE2, angiotensin-(1–7), and Mas: the other side of the coin

The renin–angiotensin system (RAS) has recently been extended by the addition of a novel axis consisting of the angiotensin-converting enzyme 2 (ACE2), the heptapeptide angiotensin (1–7) (Ang-(1–7)), and the G protein-coupled receptor Mas. ACE2 converts the vasoconstrictive and pro-oxidative peptide angiotensin II (Ang II) into Ang-(1–7) which exerts vasodilatory and antioxidative effects via its receptor Mas. Thereby, ACE2 regulates the local actions of the RAS in cardiovascular tissues and the ACE2/Ang-(1–7)/Mas axis exerts protective actions in hypertension, diabetes, and other cardiovascular disorders. Consequently, this novel RAS axis represents a promising therapeutic target for cardiovascular and metabolic diseases.     

Involvement of capsaicin-sensitive afferent nerves in the proteinase-activated receptor 2–mediated vasodilatation in the rat dura mater

Neurogenic inflammation of the dura mater encephali has been suggested to contribute to the mechanisms of meningeal nociception and blood flow regulation. Recent findings demonstrated that the rat dura mater is innervated by trigeminal capsaicin-sensitive peptidergic nociceptive afferent nerves which mediate meningeal vascular responses through activation of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The present work explored the functional significance of the capsaicin-sensitive subpopulation of dural afferent nerves via their contribution to the meningeal vascular responses evoked through activation of the proteinase-activated receptor 2 (PAR-2). The vascular responses of the dura mater were studied by laser Doppler flowmetry in a rat open cranial window preparation. Topical applications of trypsin, a PAR-2-activator, or Ser–Leu–Ile–Gly–Arg–Leu–amide (SLIGRL-NH₂), a selective PAR-2 agonist peptide, resulted in dose-dependent increases in meningeal blood flow. The SLIGRL-NH₂-induced vasodilatation was significantly reduced following capsaicin-sensitive afferent nerve defunctionalization by prior systemic capsaicin treatment and by pretreatment of the dura mater with the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP_8-37. Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) an unspecific inhibitor of nitric oxide (NO) production, but not 1-(2-trifluoromethylphenyl) imidazole (TRIM), a neuronal NO synthase inhibitor, also inhibited the vasodilator response to SLIGRL-NH₂. The vasodilator responses elicited by very low concentrations of capsaicin (10 nM) were significantly enhanced by prior application of SLIGRL-NH₂. The present findings demonstrate that activation of the PAR-2 localized on capsaicin-sensitive trigeminal nociceptive afferent nerves induces vasodilatation in the dural vascular bed by mechanisms involving NO and CGRP release. The results indicate that the PAR-2-mediated activation and sensitization of meningeal capsaicin-sensitive C-fiber nociceptors may be significantly implicated in the pathophysiology of headaches.     

CISH constrains the tuft–ILC2 circuit to set epithelial and immune tone

Innate lymphoid cells (ILCs) are tissue-resident effectors poised to activate rapidly in response to local signals such as cytokines. To preserve homeostasis, ILCs must employ multiple pathways, including tonic suppressive mechanisms, to regulate their primed state and prevent inappropriate activation and immunopathology. Such mechanisms remain incompletely characterized. Here we show that cytokine-inducible SH2-containing protein (CISH), a suppressor of cytokine signaling (SOCS) family member, is highly and constitutively expressed in type 2 innate lymphoid cells (ILC2s). Mice that lack CISH either globally or conditionally in ILC2s show increased ILC2 expansion and activation, in association with reduced expression of genes inhibiting cell-cycle progression. Augmented proliferation and activation of CISH-deficient ILC2s increases basal and inflammation-induced numbers of intestinal tuft cells and accelerates clearance of the model helminth, Nippostrongylus brasiliensis, but compromises innate control of Salmonella typhimurium. Thus, CISH constrains ILC2 activity both tonically and after perturbation, and contributes to the regulation of immunity in mucosal tissue.     

Periodontal disease and type 2 diabetes mellitus: Is the HMGB1–RAGE axis the missing link?

Periodontitis is a major chronic inflammatory disease associated with increased production of numerous proinflammatory cytokines, which leads to the destruction of the periodontal tissue and ultimately loss of teeth. Periodontitis has powerful and multiple influences on the occurrence and severity of systemic conditions and diseases, such as diabetes mellitus, cardiovascular disease and respiratory disease. Meanwhile, diabetes is associated with increased prevalence, severity and progression of periodontal disease. There is also abundant evidence showing that diabetes plays important etiological roles in periodontitis. High mobility group box 1 (HMGB1) was recently identified as a lethal mediator of severe sepsis and comprises a group of intracellular proteins that function as inflammatory cytokines when released into the extracellular milieu. From a clinical perspective, extracellular HMGB1 can cause multiple organ failure and contribute to the pathogenesis of sepsis, rheumatoid arthritis, cardiovascular disease and diabetes. We recently reported that HMGB1 expression in periodontal tissues was elevated in patients with severe periodontitis. In addition, the receptor for advanced glycation end-products (RAGE), a receptor for HMGB1, was strongly expressed in gingival tissues obtained from patients with type 2 diabetes and periodontitis compared with systemically healthy patients with chronic periodontitis patients. From these data, we hypothesize that HMGB1 might play a role in the development of diabetes-associated periodontitis.     

Emergence and evolution of the renin–angiotensin–aldosterone system

The renin-angiotensin-aldosterone system (RAAS) is not the sole, but perhaps the most important volume regulator in vertebrates. To gain insights into the function and evolution of its components, we conducted a phylogenetic analysis of its main related genes. We found that important parts of the system began to appear with primitive chordates and tunicates and that all major components were present at the divergence of bony fish, with the exception of the Mas receptor. The Mas receptor first appears after the bony-fish/tetrapod divergence. This phase of evolutionary innovation happened about 400 million years ago. We found solid evidence that angiotensinogen made its appearance in cartilage fish. The presence of several RAAS genes in organisms that lack all the components shows that these genes have had other ancestral functions outside of their current role. Our analysis underscores the utility of sequence comparisons in the study of evolution. Such analyses may provide new hypotheses as to how and why in today's population an increased activity of the RAAS frequently leads to faulty salt and volume regulation, hypertension, and cardiovascular diseases, opening up new and clinically important research areas for evolutionary medicine.     

The emerging role of the Nrf2–Keap1 signaling pathway in cancer

The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2])–Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1) signaling pathway is one of the most important cell defense and survival pathways. Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. As a result, several Nrf2 activators are currently being tested as chemopreventive compounds in clinical trials. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression. Nrf2 is aberrantly accumulated in many types of cancer, and its expression is associated with a poor prognosis in patients. In addition, Nrf2 expression is induced during the course of drug resistance. Collectively, these studies suggest that Nrf2 contributes to both intrinsic and acquired chemoresistance. This discovery has opened up a broad spectrum of research geared toward a better understanding of the role of Nrf2 in cancer. This review provides an overview of (1) the Nrf2–Keap1 signaling pathway, (2) the dual role of Nrf2 in cancer, (3) the molecular basis of Nrf2 activation in cancer cells, and (4) the challenges in the development of Nrf2-based drugs for chemoprevention and chemotherapy.