Reducing Viral Inhibition of Host Cellular Apoptosis Strengthens the Immunogenicity and Protective Efficacy of an Attenuated HSV-1 Strain

Herpes simplex virus 1(HSV-1), a member of a herpesviruses, shows a high infectivity rate of 30%–60% in populations of various ages. Some herpes simplex(HSV) vaccine candidates evaluated during the past 20 years have not shown protective efficacy against viral infection. An improved understanding of the immune profile of infected individuals and the associated mechanism is needed. HSV uses an immune evasion strategy during viral replication, and various virus-encoded proteins, such as ICP47 and Vhs, participate in this process through limiting the ability of CD8?cytotoxic T lymphocytes to recognize target cells. Other proteins, e.g., Us3 and Us5, also play a role in viral immune evasion via interfering with cellular apoptosis. In this work, to study the mechanism by which HSV-1 strain attenuation interferes with the viral immune evasion strategy, we constructed a mutant strain, M5, with deletions in the Us3 and Us5 genes. M5 was shown to induce higher neutralizing antibody titers and a stronger cellular immune response than our previously reported M3 strain,and to prevent virus infection more effectively than the M3 strain in an in vivo mouse challenge test.     

2型单纯疱疹病毒体外感染周期的研究进展

2型单纯疱疹病毒(Herpes simplex virus type 2,HSV-2)是引起生殖器疱疹的主要病原体。生殖器疱疹主要表现为生殖器和肛周皮肤黏膜疱疹或溃疡,是一种慢性、复发性、难以治愈的性传播疾病,临床治疗多以抑制病毒复制的核苷类药物阿昔洛韦及其衍生物更昔洛韦、喷昔洛韦等为主。这些药物对缓解症状、缩短病程有一定作用,但难以达到根治的目的,长期应用易产生耐药,且对其合并症基本无效。作用于HSV-2其他感染周期的药物成为人类探索的新领域。HSV-2感染宿主细胞是一个复杂的多阶段过程,包括黏附、穿入、核转运、基因组复制、衣壳组装、子代病毒释放等多个步骤,涉及多种细胞和病毒蛋白的活性。本文对HSV-2体外感染周期详细步骤及其分子机制作一综述,以期深入了解其感染机制,并为研制作用于不同感染阶段的抗HSV-2药物提供参考。     

Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.     

COMPASS核心成员Ash2l通过调控细胞周期影响神经祖细胞增殖

为探究Ash2l(absent, small, or homeotic 2-like, Ash2l)对小鼠大脑皮质神经祖细胞(neural progenitor cells, NPCs)的增殖能力和细胞周期的影响。本研究利用NPCs标志物PAX6和TBR2,检测NPCs数量和分布的改变情况。结果显示,Ash2l敲除导致NPCs数量显著减少(P<0.05),且分布紊乱。对E16.5小鼠进行在体30 min EdU标记实验,检测NPCs 增殖能力,Ash2l敲除导致30 min EdU几乎无法进入NPCs(P<0.001)。结果表示,NPCs增殖能力受到严重的影响。用细胞周期M期标志物pH3,检测大脑皮质中处于M期的NPCs分布情况,同时提取了E16.5小鼠大脑皮质蛋白质,检测细胞周期蛋白 A的表达量。Ash2l敲除的NPCs的 M期细胞核分布紊乱,G₂期标志蛋白质细胞周期蛋白 A表达量减少。利用EdU和BrdU双标记法,计算NPCs的S期长度。Ash2l敲除后的NPCs的S期长度缩短(P<0.05)。因此,Ash2l调控NPCs细胞周期进程,进而影响NPCs的增殖能力,敲除小鼠大脑皮质发育异常。本研究强调了表观遗传调控对胚胎期神经系统发育的重要作用,并对表型进行了深入探索。     

Osmotic Stress-induced Phosphorylation of H2AX by Polo-like Kinase 3 Affects Cell Cycle Progression in Human Corneal Epithelial Cells

Increased concentrations of extracellular solutes affect cell function and fate by stimulating cellular responses, such as evoking MAPK cascades, altering cell cycle progression, and causing apoptosis. Our study results here demonstrate that hyperosmotic stress induced H2AX phosphorylation (γH2AX) by an unrevealed kinase cascade involving polo-like kinase 3 (Plk3) in human corneal epithelial (HCE) cells. We found that hyperosmotic stress induced DNA-double strand breaks and increased γH2AX in HCE cells. Phosphorylation of H2AX at serine 139 was catalyzed by hyperosmotic stress-induced activation of Plk3. Plk3 directly interacted with H2AX and was colocalized with γH2AX in the nuclei of hyperosmotic stress-induced cells. Suppression of Plk3 activity by overexpression of a kinase-silencing mutant or by knocking down Plk3 mRNA effectively reduced γH2AX in hyperosmotic stress-induced cells. This was consistent with results that show γH2AX was markedly suppressed in the Plk3^−/− knock-out mouse corneal epithelial layer in response to hyperosmotic stimulation. The effect of hyperosmotic stress-activated Plk3 and increased γH2AX in cell cycle progression showed an accumulation of G₂/M phase, altered population in G₁ and S phases, and increased apoptosis. Our results for the first time reveal that hyperosmotic stress-activated Plk3 elicited γH2AX. This Plk3-mediated activation of γH2AX subsequently regulates the cell cycle progression and cell fate.     

Dynamic Organization of Microtubules and Microfilaments during Cell Cycle Progression in Higher Plant Cells

Abstract: The cytoskeleton, which mainly consists of microtubules (MTs) and actin microfilaments (MFs), plays various significant roles that are indispensable for eukaryotic viability, including determination of cell shape, cell movement, nuclear division, and cytokinesis. In animal cells, MFs appear to be of more importance than MTs, except for spindle formation in nuclear division. In contrast, higher plants have a rigid cell wall around their cells, and have thus evolved elegant systems of MTs to control the direction of cellulose microfibrils (CMFs) deposited in the cell wall, and to divide centrifugally in a physically limited space. Dynamic changes in MTs during cell cycle progression in higher plant cells have been observed over several decades, including cortical MTs (CMTs) during interphase, preprophase bands (PPBs) from late G₂ phase to prophase, spindles from prometaphase to anaphase, and phragmoplasts at telophase. The MFs also show some changes not as obvious as MT dynamics. However, questions regarding the process of formation of these arrays, and the precise mechanisms by which they fulfill their roles, remain unsolved. In this article, we present an outline of the changes in the cytoskeleton based on our studies with highly-synchronized tobacco BY-2 cells. Some candidate molecules that could play roles in cytoskeletal dynamics are discussed. We also hope to draw attention to recent attempts at visualization of cytoskeletons with molecular techniques, and to some examples of genetic approaches in this field.     

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders.     

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A₂ (TxA₂)/TxA₂ receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G₂/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G₂/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA₂/TP promotes MM cell proliferation by reducing cell delay at G₂/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.     

miR-638 Regulates Differentiation and Proliferation in Leukemic Cells by Targeting Cyclin-dependent Kinase 2

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G₁ cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway

Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21(Cip1)-dependent G(1)-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G(1) arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G(1) arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G(1) arrest.     

D609 对Neuro-2a 脑神经瘤细胞增殖和细胞周期的影响

目的:探讨小分子化合物D609对脑神经瘤细胞Neuro-2a的生长抑制及诱导细胞周期阻滞的效应,并初步研究其机制。方法:采用CCK-8法检测D609对Neuro-2a细胞的生长抑制作用;利用流式细胞术(FACS)检测D609处理对细胞周期进程的影响;利用免疫印迹实验(Western blot)检测不同浓度的D609处理后,细胞裂解液中细胞周期蛋白抑制因子p27的表达水平。结果:CCK-8的实验结果显示,加入150μmol/L D609处理72小时后,细胞生长受到明显地抑制,且伴有剂量依赖效应;流式细胞术的结果表明,D609处理使细胞周期阻滞在G0/G1期;免疫印迹的结果表明药物处理提升了p27的表达,且随药物浓度升高其表达亦增强。结论:D609可以有效地抑制Neuro-2a细胞的生长;进一步研究表明药物处理可以提升p27的表达水平并可以诱导将细胞阻滞在G0/G1期。因此,此研究将为脑神经瘤的治疗提供借鉴。     

Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL

Virologica Sinica - Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a...     

SNX-2112, a Novel Hsp90 Inhibitor,Induces G2/M Cell Cycle Arrest and Apoptosis in MCF-7 Cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.