Identification of 'Non-Proliferating' B16 Melanoma Cells Using Monoclonal Antibody (Ad203) Against the M1 Subunit of Ribonucleotide Reductase

Abstract. Ribonucleotide reductase catalyses a critical reaction in DNA synthesis. Its M1 subunit is present during all proliferative phases of the cell cycle, but apparently not in the quiescent phase Go. We have used a monoclonal antibody (AD203) directed against the M1 subunit to distinguish immunocytochemically proliferating from non-proliferating cultured B16 mouse melanoma cells during exponential growth. the presumption that AD203 unstained cells constituted a non-proliferating fraction was tested by simultaneously counting the cells that failed to incorporate bromodeoxyuridine (BrdU) into DNA during a prolonged BrdU exposure. the proportion of cells which did not incorporate BrdU was found to correlate closely with the proportion not staining with AD203 and therefore presumably lacking the M1 subunit. the respective morphological features of AD203 stained and unstained cells were found not to differ significantly.     

Identification of an HLA-A2-Restricted Epitope Peptide Derived from Hypoxia-Inducible Protein 2 (HIG2)

We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.     

In Situ IgM Production and Clonal Expansion of B-1 Cells in Peritoneal Cavity Promote Elimination of C. albicans Infection in IgH Transgenic Mice with VH Derived from a Natural Antibody

B-1 cells are innate-like cells that play important roles in host defense against infection. However, the function of B-1 cells in fungi infection remains unclear. Previously we produced IgH transgenic mice TgV_H3B4 with V_H derived from a natural antibody 3B4 that can identify C. albicans, and found that TgV_H3B4 mice were resistant to intraperitoneal (i. p.) and intravenous C. albicans infection. Most of the peritoneal cavity (PEC) B-1 cells in TgV_H3B4 mice express transgenic BCR that binds C. albicans. In the present study, we explored the response of B-1 cells to C. albicans infection by applying i. p. inoculation of fungi in TgV_H3B4 mice. We found that C. albicans was cleared more efficiently in TgV_H3B4 mice after i. p. inoculation than that of littermate control. The level of C. albicans-reactive IgM in PEC of TgV_H3B4 mice was much higher than that of control, and the number of B-1a B cells was also elevated in TgV_H3B4 mice, which was mainly due to enhanced proliferation of B-1 cells. Additionally, numbers of C. albicans-specific B cells increased greatly in TgV_H3B4 mice after C. albicans inoculation. Our data suggested that in situ IgM production and clonal expansion of B-1 cells in PEC participate in host defense against C. albicans infection.     

人高致病性禽流感病毒H5N1安徽株HA、M2蛋白双顺反子表达载体的构建及表达研究

将我国分离的人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和M2基因片段并克隆至DNA疫苗表达载体pVRC中,构建成真核表达质粒。为提高HA的表达量,按照人偏爱密码子将HA基因进行优化改造,经全基因合成后插入真核表达载体pVRC,以β-actin蛋白为内参比证明了优化后的HA蛋白表达效果明显提高。将M2基因和优化后的HA基因共同克隆入双顺反子表达载体pIRES中,获得同时表达HA或M2的双顺反子真核表达质粒;通过Western blot和间接免疫荧光检测方法,确认构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和M2。通过上述结果为进一步开展人高致病性禽流感病毒安徽株HA和M2基因的功能与致病性研究及使用表达HA和M2蛋白进行新型人用禽流感双价疫苗研发奠定基础。     

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.     

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.     

Evidence Against an Acute Inhibitory Role of nSec-1 (Munc-18) in Late Steps of Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: nSec-1 (munc-18) is a mammalian homologue of proteins implicated in constitutive exocytosis in yeast and neurotransmission in Caenorhabditis elegans and Drosophila . Mutant phenotypes in these species suggest that nSec-1 is likely to be required for neurotransmission. Various other data have been interpreted as suggesting that nSec-1 could also be a negative regulator of Ca²⁺-dependent exocytosis. We have tested this possibility by introducing exogenous nSec-1 into permeabilised chromaffin or PC12 cells and examining its effects on Ca²⁺-induced and α-soluble N -ethylmaleimide-sensitive fusion protein attachment protein-stimulated exocytosis. No effects of exogenous nSec-1 were observed in these assays. In addition, the effect of nSec-1 overexpression in transiently transfected PC12 cells on reporter growth hormone (GH) secretion was examined. Overexpression of nSec-1 resulted in a marked increase in GH production, reflected in an increase in both cell-associated and medium GH levels. The relative amounts retained in the cells were unaffected by nSec-1 overexpression, indicating that GH storage was unaffected and that the major effect was on its synthesis. In contrast, nSec-1 overexpression did not affect the proportion of GH that was released following stimulation in intact or permeabilised cells. These results suggest either that nSec-1 is already expressed at sufficient levels and remains so following permeabilisation or that nSec-1 may not be an acute inhibitory regulator of Ca²⁺-dependent exocytosis in chromaffin or PC12 cells.     

Autosomal Tubulointerstitial Kidney Disease—MUC1 Type: Differential Proteomics Suggests that Mutated MUC1 (insC) Affects Vesicular Transport in Renal Epithelial Cells

Autosomal dominant tubulointerstitial kidney disease associated to the MUC1 gene (ADTKD‐MUC1; formerly MCKD1) belongs to a heterogeneous group of rare hereditary kidney diseases that is prototypically caused by frameshift mutations in the MUC1 repeat domain. The mutant MUC1 (insC) lacks the transmembrane domaine, exhibits aberant cellular topology, and hence might gain a function during the pathological process. To get insight into potential pathomechanisms we perform differential proteomics of extracellular vesicles shed by renal epithelia into the urine of patients. The study is based on three ADTKD patients and individual controls applying iTRAQ/LC–MS/MS. A total of 796 proteins were identified across all biological and technical replicates, and 298 proteins were quantified. A proportion of 47 proteins were fold‐changed species. GO Term Enrichment analysis revealed proteins with significantly changed expression in ADTKD‐associated extracellular vesicles as vesicular transport‐associated proteins. Among these VTA1 is involved in the endosomal multivesicular body pathway associated with transport to lysosomes or export via exosomes. VTA1 is also claimed to play roles as a cofactor of the AAA ATPases VPS4A and VPS4B in the disassembly of ESCRT III. Protein interaction databases list VPS4B, CHMP2A, and IST1 as VTA1 binding partners. (Data are available via ProteomeXchange with identifier PXD008389.)     

Identification of Hsc70 as an influenza virus matrix protein (M1) binding factor involved in the virus life cycle

Influenza virus matrix protein 1 (M1) has been shown to play a crucial role in the virus replication, assembly and budding. We identified heat shock cognate protein 70 (Hsc70) as a M1 binding protein by immunoprecipitation and MALDI-TOF MS. The C terminal domain of M1 interacts with Hsc70. We found that Hsc70 does not correlate with the transport of M1 to the nucleus, however, it does inhibit the nuclear export of M1 and NP, thus resulting in the inhibition of viral production. This is the first demonstration that Hsc70 is directly associated with M1 and therefore is required for viral production.     

A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study

There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells.     

Antibody from Patients with Acute Human Immunodeficiency Virus (HIV) Infection Inhibits Primary Strains of HIV Type 1 in the Presence of Natural-Killer Effector Cells

The partial control of viremia during acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by an HIV-1-specific cytotoxic T-lymphocyte (CTL) response and an absent or infrequent neutralizing antibody response. The control of HIV-1 viremia has thus been attributed primarily, if not exclusively, to CTL activity. In this study, the role of antibody in controlling viremia was investigated by measuring the ability of plasma or immunoglobulin G from acutely infected patients to inhibit primary strains of HIV-1 in the presence of natural-killer (NK) effector cells. Antibody that inhibits virus when combined with effector cells was present in the majority of patients within days or weeks after onset of symptoms of acute infection. Furthermore, the magnitude of this effector cell-mediated antiviral antibody response was inversely associated with plasma viremia level, and both autologous and heterologous HIV-1 strains were inhibited. Finally, antibody from acutely infected patients likely reduced HIV-1 yield in vitro both by mediating effector cell lysis of target cells expressing HIV-1 glycoproteins and by augmenting the release of beta-chemokines from NK cells. HIV-1-specific antibody may be an important contributor to the early control of HIV viremia.