Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity / 生物工程学报

Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein, and its inhibitory activity on tumor cells and angiogenesis were assessed. luffin-α-NGR fusion gene was obtained by PCR amplification. The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR. The plasmid was transformed into E. coli BL21, and the target protein was isolated and purified by GST affinity chromatography. The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained, and the optimal soluble expression of the target protein was achieved under the conditions of 16 ℃, 0.5 mmol/L IPTG after 16 h induction. SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa. Subsequently, the recombinant protein was de-tagged by precision protease digestion. The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α. The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization. The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.

Caracterização do papel de Nop53 na montagem da subunidade ribossomal maior em Saccharomyces cerevisiae / Characterization of the role of Nop53 in the assembly of the large ribosomal subunit in Saccharomyces cerevisiae

Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2

Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle

Protective effects of bicistronic DNA vaccines of herpes simplex virus type 2 glycoprotein D and adjuvant CCL28 on mice / 中华微生物学和免疫学杂志

Objective:To study the protective effects of bicistronic DNA vaccines carrying herpes simplex virus type 2 glycoprotein D (HSV-2 gD) and adjuvant CCL28 sequences that were connected by internal ribosome entry site (IRES) sequence in mouse model.Methods:The recombinant DNA vaccines, pgD-IRES-CCL28 and pCCL28-IRES-gD, encoding HSV-2 gD and adjuvant CCL28 were constructed with IRES sequence. After verified by sequencing, they were intramuscularly injected twice into BALB/c mice. Serum samples and vaginal lavage fluids were collected regularly. Splenocytes, mesenteric lymph node cells and rectal mucosa tissues were separated and collected. The titers of antigen-specific antibodies in immunized mice were analyzed with end-point ELISA. In vitro neutralization assay was used to measure neutralizing antibody titers in serum and vaginal lavage fluid after vaccination and virus challenge. CCL28-responsive immune cells in splenocytes, mesenteric lymph node cells and rectal tissues were detected by chemotaxis experiment and immunohistochemical staining. The protective effects of the bicistronic DNA vaccines were evaluated by fluorescent quantitative PCR, weighing and disease severity assessment. Humoral and cellular immune responses induced by the bicistronic DNA vaccines and their efficacy in immunoprotection were analyzed by comparing with pgD+ pCCL28 group. Results:IgG titers in serum samples and IgA antibody titers in vaginal lavage fluids of mice immunized with pCCL28-IRES-gD were similar to those in pgD+ pCCL28 group. The neutralizing ability of antibodies, the number of rectal mucosal IgA+ plasma cells and CCL28-responsive immune cells in mucosal tissues were increased in pCCL28-IRES-gD group. Serum neutralizing antibodies were not produced immediately in the mice challenged with HSV-2, but no weight loss, disease symptoms or death was observed. However, pgD+ pcDNA3.1 and pgD-IRES-CCL28 were ineffective against HSV-2 infection in mice.Conclusions:The recombinant bicistronic DNA vaccine of pCCL28-IRES-gD could induce stronger mucosal immune response in mice and provide better protective effects.

Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques / 生物工程学报

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.

Mechanism of ribosome biogenesis and exercise adaptation of skeletal muscle hypertrophy / 中国组织工程研究

BACKGROUND: Research evidence shows that skeletal muscle contractile activity can induce ribosomal biogenesis, which plays an important role in the control of skeletal muscle mass. OBJECTIVE: To review the main mechanism of ribosome biogenesis in skeletal muscle hypertrophy, upstream regulatory signals of ribosomal biogenesis in skeletal muscle, and effect of exercise on ribosomal biogenesis, and to explore the ribosome biogenesis mechanism of exercise-induced skeletal muscle hypertrophy. METHODS: Relevant studies about exercise, skeletal muscle hypertrophy and ribosome biogenesis in CNKI, Wanfang, and PubMed databases were searched. The key words were “exercise, resistance training, skeletal muscle hypertrophy, protein synthesis, ribosome biogenesis” in English and Chinese. Relevant literatures published from 1999 to 2019 were searched and screened according to inclusion and exclusion criteria. RESULTS AND CONCLUSION: (1) Ribosome biogenesis as a main source of translational capacity plays an important role in muscle growth. (2) A single bout of resistance exercise can promote the ribosome biogenesis. However, cumulative bouts of resistance exercise eventually lead to the accumulation of mature rRNAs, leading to increased concentration of total RNA, which promote the growth of skeletal muscle. (3) Ribosome biogenesis may be the key molecular mechanism for the regulation of skeletal muscle hypertrophy induced by resistance training. (4) Moderate-volume resistance training led to adaptations to resistance training. This hypertrophy was associated with volume-dependent regulation of total RNA. This suggests that ribosomal biogenesis regulates the dose-effect relationship between training volume and muscle hypertrophy.

Research progress of Phytolaccae Radix / 中草药

This paper summarizes the herbal textual research, authenticity identification, processing, composition and pharmacology, clinical studies, industry and agriculture resources development of Phytolaccae Radix based on the ancient herbology works and references. The herbal textual research showed that Phytolaccae Radix had different names like Shanglu or Danglu or Zhanglu. The effects are external application of carbuncle, swelling and sore toxin, internal administration of diuretic and hydroncus. The main producing area is transferred from northwest to southeast. The root has the abnormal structure of concentric ring, which is different from the eight kinds of common adulterants. It is mainly processed with vinegar to reduce toxicity and increase efficacy. Triterpenoid saponins, polysaccharides and pokeweed antiviral proteins (PAPs) are the main effective components of Phytolaccae Radix, which have the functions of diuresis and diarrhea, improving inflammation of kidney, liver, respiratory tract and neuroinflammatory diseases, as well as antibacterial, antiviral, antitumor and other pharmacological activities. It is mainly used for the treatment of cirrhosis ascites, nephrotic syndrome and external application for the treatment of constipation clinically. Phytolaccae Radix has important application value in plant disease resistance, insect resistance and environmental restoration. The clinical efficacy of Phytolaccae Radix is clear and it has made important progress in the research of components, pharmacology, industrial and agricultural resources development, which provides support for the rational development and comprehensive utilization of it.

Novel small molecule retrograde transport blocker confers post-exposure protection against ricin intoxication

Ricin is a highly toxic type 2 ribosome-inactivating protein (RIP) which is extracted from the seeds of castor beans. Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far. In this study, by structural modification of a retrograde transport blocker Retro-2, a series of novel compounds were obtained. The primary screen revealed that compound has an improved anti-ricin activity compare to positive control. pre-exposure evaluation in Madin-Darby Canine Kidney (MDCK) cells demonstrated that is a powerful anti-ricin compound with an EC of 41.05 nmol/L against one LC (lethal concentration, 5.56 ng/mL) of ricin. Further studies surprisingly indicated that confers post-exposure activity against ricin intoxication. An study showed that 1 h post-exposure administration of can improve the survival rate as well as delay the death of ricin-intoxicated mice. A drug combination of with monoclonal antibody mAb4C13 rescued mice from one LD (lethal dose) ricin challenge and the survival rate of tested animals is 100%. These results represent, for the first time, indication that small molecule retrograde transport blocker confers both and post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.

Effect of key notes of TCA cycle on L-glutamate production / 生物工程学报

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Promotion of self-nucleic acid fragments on the assembly of foot-and-mouth disease virus-like particles / 生物工程学报

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.

Estudos funcionais e biológicos associados à interação de DUSP3 com a proteína HNRNPC1/C2 / Functional and biological studies associated to DUSP3 and HNRNPC1/C2 protein inte ractions

A regulação da fosforilação/desfosforilação das proteínas é o eixo central de muitas cascatas de sinalização. A fosfatase DUSP3, constituída apenas por um único domínio catalítico, desempenha papéis fundamentais na proliferação e senescência celular. Nas células HeLa, submetidas ao estresse genotóxico, o DUSP3 interage fisicamente com as proteínas HNRNPC, mas o efeito dessa função molecular ainda é desconhecido. Aqui demostramos que a ausência de DUPS3 mantem a proteína HNRNPC1/C2 num estado hiperfosforilado. Para entender melhor o envolvimento da interação DUSP3-HNRNPC nas funções biológicas da HNRNPC1/C2, foram estudadas células de fibroblasto deficientes de DUSP3. Foi analisado o efeito da deficiência de DUSP3 na biogênese dos ribossomos através do ensaio de perfil de polirribossomos e quantificação dos rRNAs com RT-qPCR. Os resultados mostraram que a deficiência de DUSP3 não afeta a maturação das subunidades ribossômicas, mas teria um impacto na transcrição dos pré-rRNAs e no acumulo das espécies 47S/45S. A expressado de genes contendo sequencias IRES foi analisado através do RT-qPCR e sua tradução ao longo do ciclo e em condições de estresse. Da expressão, não existe nenhuma diferença nos níveis de transcrição dos genes c-myc e xiap nas células normais e deficientes de DUSP3 em condições basais. Embora a síntese destas proteínas é maior nas células deficientes, mantendo um nível maior de tradução ao longo de todo o ciclo. Sob condições de estresse, esta duas proteínas sempre mantem uma maior expressão nas células Knockdown para DUSP3. Neste trabalho também foi estabelecido a presença de DUSP3 nos complexos da subunidade 40S, através do analise das frações obtidas do ensaio de polirribossomos e interação in vitro (Co-IP). A presença de DUSP3 nas subunidades 40S, os monossomas 80S e polissomos poderia ser através da interação direta com proteínas que possuem um domínio RRM e seria dependente dos complexos formados pelas proteínas e seus RNAs alvos. Aqui mostramos a interação in vitro de DUSP3 com a proteína PABP (com quatro domínios RRM), proteína que tem um papel importante na manutenção da taxa global de tradução, esta interação é enfraquecida na ausência de RNAs. A deficiência de DUSP3 também teria um impacto na interação das proteínas HNRNPC1/C2 e P53 in vitro. A ausência de DUSP3 diminui a interação HNRNPC-P53 através da hiperfosforilação da proteina HNRNPC1/C2. A perda desta interação, aumentaria os níveis da proteína P53 na célula deficiente de DUSP3 e poderia gerar parada no ciclo celular. Através de ensaios de imunofluorescência, se observo uma maior taxa de transcrição global na célula deficiente de DUSP3. Por fim, aqui demostramos que a interação direta de DUSP3 e HNRNPC1/C2 vai permitir a regulação das funções biológicas desta proteína, e a ausência de DUSP3 vai ter efeitos pleiotrópicos na homeostase da célula

inglêsProtein phosphorylation/dephosphorylation regulation is a central axis of many signaling cascades. DUSP3 phosphatase, consisting only of a single catalytic domain, plays key roles in cell proliferation and senescence. In HeLa cells subjected to genotoxic stress, DUSP3 physically interacts with HNRNPC proteins, but the effect of this molecular function is still unknown. Here we demonstrate that the absence of DUPS3 keeps the HNRNPC1/C2 proteins in a hyperphosphorylated state. To better understand the involvement of DUSP3- HNRNPC interaction on the biological functions of HNRNPC1/C2, DUSP3 deficient fibroblast cells were studied. The effect of DUSP3 deficiency on ribosome biogenesis was analyzed by polyribosome profile assay and RT-qPCR for rRNA quantification. The results showed that DUSP3 deficiency does not affect ribosomal subunit maturation, but would have an impact on transcription of pre-rRNAs and accumulation of 47S / 45S species. The expression of genes containing IRES sequences was analyzed by RT-qPCR and their translation throughout the cycle and under stress conditions. From expression, there is no difference in transcriptional levels of c-myc and xiap genes in normal and DUSP3 deficient cells under basal conditions. Although, the synthesis of these proteins is higher in deficient cells and these maintain a higher level of translation throughout the cell cycle. Under stress conditions, these two proteins always maintain higher expression in Knockdown cells for DUSP3. In this work, the presence of DUSP3 in the 40S ribosomal subunit complexes was also established by analyzing the fractions obtained from the polyribosome assay and in vitro interaction (CoIP). The presence of DUSP3 in the 40S subunits, 80S monosomes and polysomes could be through direct interaction with proteins that have an RRM domain and would be dependent on the complexes formed by the proteins and their target RNAs. Here we show the in vitro interaction of DUSP3 with PABP protein (with four RRM domains), a protein that plays an important role in maintaining the overall translation rate, this interaction is weakened in the absence of RNAs. DUSP3 deficiency would also have an impact on the interaction of HNRNPC1/C2 and P53 proteins in vitro. The absence of DUSP3 decreases HNRNPC-P53 interaction through hyperphosphorylation of the HNRNPC1/C2 proteins. Loss of this interaction would increase P53 protein levels in the DUSP3 deficient cell and could lead to cell cycle arrest. Through immunofluorescence assays, a higher overall transcription rate is observed in the DUSP3 deficient cell. Finally, we demonstrate that the direct interaction of DUSP3 and HNRNPC1/C2 will allow the regulation of the biological functions of this protein, and the absence of DUSP3 will have pleiotropic effects on cell homeostasis

Effects and mechanism of Momordica charantia MAP30 on migration of bladder cancer / 中国医师杂志

Objective To study the effect and mechanism of Momordica anti-HIV protein of 30 ku (MAP30) on the migration of bladder cancer.Methods The IC50 of human bladder cancer 5637 and T24 cells was calculated by methyl thiazolyl tetrazolium (MTT) method.The migration ability of these two cells was evaluated by scratch migration test and Transwell cell migration test.The expression of migrating proteins such as matrix metalloproteinases (MMPs) and adhesion molecule N-cadherin were compared by Western blot.Results Scratch migration test:there were significant differences in migration rates of 5637 cells at 8 h and 22 h (P ＜ 0.05).There were significant differences in migration rates of T24 cells at 22 h (P ＜ 0.05),but no significant differences in migration rates at 8 h (P ＞ 0.05).The expression of Vimentin,Fibronectin,MMP-2,MMP-9 and N-Cadherin in 5637 cells and T24 cells of human bladder cancer decreased significantly after adding MAP30.The E-Cadherin expression in human bladder cancer 5637 cells were decreased,but no target band was detected in human bladder cancer T24 cells.Conclusions The ribosome-inactivating protein MAP30 can effectively inhibit the migration of human bladder cancer 5637 and T24 cells by inhibiting the EMT pathway and inhibiting the expression of MMPs.

Expression and clinical significance of ribosome-binding protein 1 in esophageal carcinoma / 国际肿瘤学杂志

Objective@#To investigate the expression and clinical significance of ribosome-binding protein 1 (RRBP1) in esophageal carcinoma.@*Methods@#A total of 120 esophageal carcinoma patients admitted to Yulin First Hospital of Shaanxi Province from January 2010 to December 2014 were enrolled in this study. RRBP1 expression was detected in esophageal carcinoma and matched adjacent normal tissues by real-time quantitative PCR (qRT-PCR), Western blotting and immunohistochemical staining. The relationships of RRBP1 expression with clinicopathological futures and prognosis were statistically analyzed.@*Results@#RRBP1 expression level was significantly higher in esophageal carcinoma tissues compared with matched adjacent normal tissues. In the mRNA level, the relative quantitative expression in tumor tissues was 3.5±1.3, and 1.0±0.3 in adjacent normal tissues, with a significant difference (t=19.130, P<0.001). In the protein level, the relative quantitative expression in tumor tissues was 1.0±0.4, and 0.3±0.1 in adjacent normal tissues, with a significant difference (t=4.225, P<0.001). The immunohistochemical results showed that RRBP1 high expression was found in 71 (59.2%) tumor tissues, and the proportion dropped to 11.7% (14/120) in adjacent normal tissues, with a significant difference (χ2=59.182, P<0.001). In addition, RRBP1 expression was correlated with lymph node metastasis and TNM stage (χ2=10.631, P=0.001; χ2=31.212, P<0.001). Survival analysis revealed that RRBP1 expression, lymph node metastasis and TNM stage were significantly correlated with patients′ prognosis (χ2=9.455, P=0.006; χ2=14.542, P<0.001; χ2=11.987, P<0.001). Cox regression analysis showed that high expression of RRBP1 (RR=2.441, 95%CI: 1.267-4.702, P=0.008), lymph node metastasis (RR=4.024, 95%CI: 2.180-7.424, P<0.001) and high TNM stage (RR=3.054, 95%CI: 1.452-6.421, P=0.003) were the risk factors for poor prognosis of esophageal carcinoma patients.@*Conclusion@#RRBP1 is highly expressed in esophageal carcinoma and can serve as a potential biomarker to predict patients′ prognosis.

Pathogenic genes and clinical therapeutic strategies for Treacher Collins syndrome / 华西口腔医学杂志

Treacher Collins syndrome is a congenital craniofacial malformation with autosomal dominant inheritance as the main genetic pattern. In this condition, the biosynthesis of ribosomes in neural crest cells and neuroepithelial cells is blocked and the number of neural crest cells that migrate to the craniofacial region decreases, causing first and second branchial arch dysplasia. Definite causative genes include treacle ribosome biogenesis factor 1 (tcof1), RNA polymerase Ⅰ and Ⅲ subunit C (polr1c), and RNA polymerase Ⅰ and Ⅲ subunit D (polr1d). This paper provides a review of research of three major patho-genic genes, pathogenesis, phenotypic research, prevention, and treatment of the syndrome.

Progress in roles and mechanisms of deacetylase SIRT7 / 生物工程学报

As a member of the Sirtuins family in mammals, SIRT7 locates in nucleus and is a highly specific H3K18Ac (acetylated lysine 18 of histone H3) deacetylase. Recent studies showed that SIRT7 could participate in the ribosomal RNA transcription, cell metabolism, cell stress and DNA damage repair through various signaling pathways. In addition, SIRT7 is also closely related with aging, heart disease and fatty liver. In particular, SIRT7 plays important roles in the regulation of initiation and development of various tumors, such as liver cancer, gastric cancer, breast cancer, bladder cancer, colorectal cancer, and head/neck squamous cell carcinoma. This review describes the cellular and molecular functions of SIRT7, and systematically summarizes recent progress of SIRT7 in human disease.

Expression and clinical significance of ribosome-binding protein 1 in esophageal carcinoma / 国际肿瘤学杂志

Objective To investigate the expression and clinical significance of ribosome-binding pro-tein 1 (RRBP1)in esophageal carcinoma. Methods A total of 120 esophageal carcinoma patients admitted to Yulin First Hospital of Shaanxi Province from January 2010 to December 2014 were enrolled in this study. RRBP1 expression was detected in esophageal carcinoma and matched adjacent normal tissues by real-time quantitative PCR (qRT-PCR),Western blotting and immunohistochemical staining. The relationships of RRBP1 expression with clinicopathological futures and prognosis were statistically analyzed. Results RRBP1 expression level was significantly higher in esophageal carcinoma tissues compared with matched adjacent nor-mal tissues. In the mRNA level,the relative quantitative expression in tumor tissues was 3. 5 ± 1. 3,and 1. 0 ± 0. 3 in adjacent normal tissues,with a significant difference (t = 19. 130,P < 0. 001). In the protein level,the relative quantitative expression in tumor tissues was 1. 0 ± 0. 4,and 0. 3 ± 0. 1 in adjacent normal tissues,with a significant difference (t = 4. 225,P < 0. 001). The immunohistochemical results showed that RRBP1 high expression was found in 71 (59. 2％)tumor tissues,and the proportion dropped to 11. 7％ (14 / 120)in adja-cent normal tissues,with a significant difference (χ2 = 59. 182,P < 0. 001). In addition,RRBP1 expression was correlated with lymph node metastasis and TNM stage (χ2 = 10. 631,P = 0. 001;χ2 = 31. 212,P <0. 001). Survival analysis revealed that RRBP1 expression,lymph node metastasis and TNM stage were signifi-cantly correlated with patients' prognosis (χ2 = 9. 455,P = 0. 006;χ2 = 14. 542,P < 0. 001;χ2 = 11. 987,P <0. 001). Cox regression analysis showed that high expression of RRBP1 (RR = 2. 441,95％ CI:1. 267-4. 702,P = 0. 008),lymph node metastasis (RR = 4. 024,95％ CI:2. 180-7. 424,P < 0. 001)and high TNM stage (RR = 3. 054,95％ CI:1. 452-6. 421,P = 0. 003)were the risk factors for poor prognosis of esophageal carci-noma patients. Conclusion RRBP1 is highly expressed in esophageal carcinoma and can serve as a potential biomarker to predict patients' prognosis.

Effects of ionizing irradiation on ribosome signaling pathway in spleen tissues of mice / 中国药理学通报

Aim To clarify the damage mechanisms of ionizing radiation on mouse spleen tissues and analyse differential protein expression in spleen tissues of mice exposed to ionizing radiation injury and normal mice from the perspective of comparative proteomics, in order to analyse the signaling pathways involved in differential proteins. Methods The mouse ionizing radiation injury model was established by 5 Gy 60Co-y ray, and the mouse spleen tissue was extracted in the ionizing radiation group and normal group, then ITRAQ combined with LC-MS/MS detection technology were used to screen differentially expressed proteins of spleen tissues from ionizing radiation group and normal group. Results A total of 17 biological signaling pathways were identified by KEGG pathway enrichment analysis(P <0. 0 5) , in which 37 differential expressed proteins were enriched in the ribosome signaling pathway pathway , including 36 differential expressed proteins downregulated and one differential expressed protein up-regulated. These pathway involved several multiple subunit proteins, such as ribosome proteins, 60S ribosome proteins and 40S ribosome proteins, which were mainly involved in biological processes, such as translation of proteins, structural composition of ribosome and so on. Conclusions Ionizing radiation causes 37 differential proteins involved in ribosome signaling pathway of mouse spleen tissues, including 36 differential expressed proteins down-regulated and one differential expressed protein up-regulated. The disturbance of protein synthesis in spleen tissues is an important mechanism of ionizing radiation injury.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Identification of anti-Gram-negative bacteria agents targeting the interaction between ribosomal proteins L12 and L10

Gram-negative bacteria have become the main pathogens and cause serious clinical problems with increased morbidity and mortality. However, the slow discovery of new antimicrobial agents is unable to meet the need for the treatment of bacterial infections caused by drug-resistant strains. The interaction of L12 and L10 is essential for ribosomal function and protein synthesis. In this study, a yeast two-hybrid system was established to successfully detect the interaction between L12 and L10 proteins from gram-negative bacteria , which allows us to screen compounds that specifically disrupt this interaction. With this system, we identified two compounds IMB-84 and IMB-87 that block L12-L10 interaction and show bactericidal activity against . We used glutathione--transferase (GST) pull-down and surface plasmon resonance (SPR) assays to demonstrate that these compounds disrupt L12-L10 interaction and the target of compounds was further confirmed by the overexpression of target proteins. Moreover, protein synthesis and elongation factor G-dependent GTPase activities are inhibited by two compounds. Therefore, we have identified two antibacterial agents that disrupt L12-L10 interaction by using yeast two-hybrid system.

Effect of silencing RRS1 gene on biological function of breast cancer BT549 cells / 肿瘤

Objective: To investigate the effects of silencing the regulator of ribosome synthesis 1 (RRS1) gene expression on proliferation, apoptosis, migration and invasion abilities of breast cancer BT549 cells, and to explore the possible mechanism. Methods: The expression levels of RRS1 mRNA and protein in breast cancer BT549 cells and normal mammary gland HMEC cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The lentivirus expression vector carrying RRS1-shRNA was constructed and transfected into BT549 cells, while the empty vector was used as the control. The silencing efficiency of RRS1 gene was identified by real-time fluorescent quantitative PCR and Western blotting, respectively. Then the proliferation, cell cycle, apoptosis, migration and invasion abilities of BT549 cells transfected with RRS1-shRNA were detected by MTT, FCM, DAPI staining and Transwell chamber assay, respectively. The expression levels of apoptosis-related proteins p53 and mouse double minute 2 homolog (MDM2) in BT549 cells transfected with RRS1-shRNA were detected by Western blotting. Results: The expression levels of RRS1 mRNA and protein in BT549 cells were significantly higher than those in the normal mammary gland HMEC cells (both P < 0.01). After transfection with RRS1-shRNA, the expression levels of RRS1 mRNA and protein in BT549 cells were significantly down-regulated as compared with the control group (both P < 0.01). After RRS1 gene silencing, the cell viability was significantly decreased (P < 0.01), the cell cycle was arrested at G2 phase (P < 0.01), the early apoptosis rate was significantly increased (P < 0.05), while the migration and invasion abilities were significantly decreased (both P < 0.05). The expression level of apoptosis-associated p53 protein was significantly up-regulated (P < 0.05), but the expression level of MDM2 protein was significantly down-regulated (P < 0.05) in BT549 cells after transfection with RRS1-shRNA. Conclusion: The RRS1 gene was highly expressed in breast cancer BT549 cells. RRS1, as a novel breast cancer related gene, maybe play an important role in the proliferation, apoptosis, migration and invasion of breast cancer cells.

In vitro-engineered non-antibody protein therapeutics

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, β-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.