Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis

Herpes simplex virus‐1 (HSV‐1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin‐dependent endocytosis plays a major role in this process. Dominant‐negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin‐dependent and ‐independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non‐infectious HSV‐1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein‐sorting event during HSV‐1 envelopment.      

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Herpes simplex virus type 1 dysregulates anti‐fungal defenses preventing monocyte activation and downregulating toll‐like receptor‐2

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP‐1 monocytic cell line is first infected with HSV‐1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV‐1‐infected THP‐1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL‐12 production was also observed. Cytofluorimetric analysis showed that HSV‐1‐infected monocytes exhibit: (i) downregulated TLR‐2 and TLR‐4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV‐1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte‐mediated anti‐Candida response; HSV‐1 induced apoptosis and necrosis of monocytes further contribute to this impairment.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibition suppress mantle cell lymphoma's cyclin D1

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells’ proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2‐oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down‐regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9‐based loss‐of‐function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1‐FOXO3A pathway and that chelation‐ and 2‐oxoglutarate competition‐mediated down‐regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron‐ and 2‐oxoglutarate‐dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibitors as a novel therapy of MCL.     

Vaccine induced HSV 1 antibodies fail to correlate with protection against HSV 2 in guinea pigs

Abstract Guinea pigs immunised with HSV 1 subunit vaccine were bled, and subsequently challenged intravaginally with HSV 2. The clinical response to virus challenge was quantified, and correlations were sought between clinical score and virus-specific serum antibody titre in functional and binding assays (ELISA, neutralization, complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity). No significant relationships were found, and it was concluded that reactivity in the serological assays chosen did not correlate with protection against HSV 2 genital infection in vaccinated female guinea pigs.     

Field evaluation on the synergistic attractiveness of 2‐(1‐undecyloxy)‐1‐ethanol and ipsenol to Monochamus saltuarius

To develop an optimal attractant for Monochamus saltuarius (Gebler) (Coleoptera: Cerambycidae), the synergistic effects of a few potential attractants (ethanol and α‐pinene as host‐plant volatiles, and ipsenol and ipsdienol as bark beetle pheromones) were tested in a pine forest combined with 2‐(1‐undecyloxy)‐1‐ethanol (monochamol), the aggregation pheromone of Monochamus species, for two consecutive years, 2014 and 2015. Total number of catches was 65 and 33 in 2014 and 2015, respectively. Ethanol or ethanol + monochamol (a base blend) were not attractive to M. saltuarius with no difference from the control. Addition of α‐pinene and ipsdienol to the base blend did not significantly increase catches. However, ipsenol was significantly synergistic to the base blend in attracting M. saltuarius in 2014, and the blend (ipsenol + base blend) attracted meaningfully higher numbers of M. saltuarius in 2015. Our study illustrates the potential for monochamol and ipsenol baits for monitoring and trapping of M. saltuarius in the field.     

The evaluation of acute toxicity,antimicrobial activity of 1‐phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole,and binding to human serum albumin

1‐Phenyl‐5‐p‐tolyl‐1H‐1, 2, 3‐triazole (PPTA) was a synthesized compound. The result of acute toxicities to mice of PPTA by intragastric administration indicated that PPTA did not produce any significant acute toxic effect on Kunming strain mice. It exhibited the various potent inhibitory activities against two kinds of bananas pathogenic bacteria, black sigatoka and freckle, when compared with that of control drugs and the inhibitory rates were up to 64.14% and 43.46%, respectively, with the same concentration of 7.06 mM. The interaction of PPTA with human serum albumin (HSA) was studied using fluorescence polarization, absorption spectra, 3D fluorescence, and synchronous spectra in combination with quantum chemistry and molecular modeling. Multiple modes of interaction between PPTA and HSA were suggested to stabilize the PPTA–HSA complex, based on thermodynamic data and molecular modeling. Binding of PPTA to HSA induced perturbation in the microenvironment around HSA as well as secondary structural changes in the protein.     

Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer

Long non‐coding RNAs (lncRNAs) have been illustrated to function as important regulators in carcinogenesis and cancer progression. However, the roles of lncRNA NNT‐AS1 in gastric cancer remain unclear. In the present study, we investigate the biological role of NNT‐AS1 in gastric cancer tumorigenesis. Results revealed that NNT‐AS1 expression level was significantly up‐regulated in GC tissue and cell lines compared with adjacent normal tissue and normal cell lines. The ectopic overexpression of NNT‐AS1 indicated the poor prognosis of GC patients. In vitro experiments validated that NNT‐AS1 knockdown suppressed the proliferation and invasion ability and induced the GC cell cycle progression arrest at G0/G1 phase. In vivo xenograft assay, NNT‐AS1 silencing decreased the tumour growth of GC cells. Bioinformatics online program predicted that miR‐424 targeted the 3′‐UTR of NNT‐AS1. Luciferase reporter assay, RNA‐immunoprecipitation (RIP) and RNA pull‐down assay validated the molecular binding within NNT‐AS1 and miR‐424, therefore jointly forming the RNA‐induced silencing complex (RISC). Moreover, E2F1 was verified to act as the target gene of NNT‐AS1/miR‐424, indicating the NNT‐AS1/miR‐424/E2F1 axis. In conclusion, our study indicates that NNT‐AS1 sponges miR‐424/E2F1 to facilitate GC tumorigenesis and cycle progress, revealing the oncogenic role of NNT‐AS1 for GC.     

Aldehyde dehydrogenase 2 activation ameliorates CCl4‐induced chronic liver fibrosis in mice by up‐regulating Nrf2/HO‐1 antioxidant pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is critical in the pathogenesis of alcoholic liver cirrhosis. However, the effect of ALHD2 on liver fibrosis remains to be further elucidated. This study aimed to demonstrate whether ALDH2 regulates carbon tetrachloride (CCl₄)‐induced liver fibrosis and to investigate the efficacy of Alda‐1, a specific activator of ALDH2, on attenuating liver fibrosis. ALDH2 expression was increased after chronic CCl₄ exposure. ALDH2 deficiency accentuated CCl₄‐induced liver fibrosis in mice, accompanied by increased expression of collagen 1α1, α‐SMA and TIMP‐1. Moreover, ALDH2 knockout triggered more ROS generation, hepatocyte apoptosis and impaired mitophagy after CCl₄ treatment. In cultured HSC‐T6 cells, ALDH2 knockdown by transfecting with lentivirus vector increased ROS generation and α‐SMA expression in an in vitro hepatocyte fibrosis model using TGF‐β1. ALDH2 overexpression by lentivirus or activation by Alda‐1 administration partly reversed the effect of TGF‐β1, whereas ALDH2 knockdown totally blocked the protective effect of Alda‐1. Furthermore, Alda‐1 administration protected against liver fibrosis in vivo, which might be mediated through up‐regulation of Nrf2/HO‐1 cascade and activation of Parkin‐related mitophagy. These findings indicate that ALDH2 deficiency aggravated CCl₄‐induced hepatic fibrosis through ROS overproduction, increased apoptosis and mitochondrial damage, whereas ALDH2 activation through Alda‐1 administration alleviated hepatic fibrosis partly through activation of the Nrf2/HO‐1 antioxidant pathway and Parkin‐related mitophagy, which indicate ALDH2 as a promising anti‐fibrotic target and Alda‐1 as a potential therapeutic agent in treating CCl₄‐induced liver fibrosis.     

Endosome maturation factors Rabenosyn‐5/VPS45 and caveolin‐1 regulate ciliary membrane and polycystin‐2 homeostasis

Primary cilium structure and function relies on control of ciliary membrane homeostasis, regulated by membrane trafficking processes that deliver and retrieve ciliary components at the periciliary membrane. However, the molecular mechanisms controlling ciliary membrane establishment and maintenance, especially in relation to endocytosis, remain poorly understood. Here, using Caenorhabditis elegans, we describe closely linked functions for early endosome (EE) maturation factors RABS‐5 (Rabenosyn‐5) and VPS‐45 (VPS45) in regulating cilium length and morphology, ciliary and periciliary membrane volume, and ciliary signalling‐related sensory behaviour. We demonstrate that RABS‐5 and VPS‐45 control periciliary vesicle number and levels of select EE/endocytic markers (WDFY‐2, CAV‐1) and the ciliopathy membrane receptor PKD‐2 (polycystin‐2). Moreover, we show that CAV‐1 (caveolin‐1) also controls PKD‐2 ciliary levels and associated sensory behaviour. These data link RABS‐5 and VPS‐45 ciliary functions to the processing of periciliary‐derived endocytic vesicles and regulation of ciliary membrane homeostasis. Our findings also provide insight into the regulation of PKD‐2 ciliary levels via integrated endosomal sorting and CAV‐1‐mediated endocytosis.     

Glycosylation of alpha-1-proteinase inhibitor and haptoglobin in ovarian cancer: evidence for two different mechanisms

The change in glycosylation of the two acute-phase proteins, alpha-1-proteinase inhibitor (API) and haptoglobin (Hp), in progressive ovarian cancer is different. This has been shown by monosaccharide analysis and lectin-binding studies of proteins purified from serum. In the glycan chains of API, there is decreased branching (more biantennary chains), less branches ending in alpha 2-3 sialic acid, more branches ending in alpha 2-6 sialic acid and more fucose, probably linked alpha 1-6 to the core region. On the other hand, Hp shows increased branching (more triantennary chains), more branches ending in alpha 2-3 sialic acid, less branches ending in alpha 2-6 sialic acid, and more fucose, probably in the alpha 1-3 linkage at the end of the chains. This is surprising because API and Hp are thought to be glycosylated by a common pathway in the liver. We have also shown that the fucose-specific lectin,lotus tetragonolobus, extracts abnormal forms of both Hp and API in ovarian cancer, but the expression of this Hp is related to tumour burden and the expression of this API is related to lack of response to therapy. It is suggested that this difference in the behaviour of API and Hp in ovarian cancer may be associated with the different changes in their glycosylation. Of the many mechanisms that could explain these findings, a likely one is that a pathological process is removing API with triantennary chains from the circulation. In addition to their normal roles (API-enzyme inhibitor and Hp-transport protein) these proteins are reported to have many other effects in biological systems, such as immunosuppression. As correct glycosylation of API and Hp is required for their normal stability/activity, changes in glycosylation could affect their functions in ovarian cancer and these modifications could alter the course of the disease.