Study on expression of the guanylate binding protein 5 and ArfGAP with SH3 domain ankyrin repeat and PH domain 1 genes among pulmonary tuberculosis patients and populations with latent tuberculosis infection / 中国医师进修杂志

Objective To investigate the expression of guanylate binding protein 5 (GBP5) and ArfGAP with SH3 domain ankyrin repeat and PH domain 1 (ASAP1) genes in pulmonary tuberculosis patients and tuberculosis latent population. Methods Forty pulmonary tuberculosis patients (pulmonary tuberculosis group), 40 latent tuberculosis infection patients (latent tuberculosis infection group) and 40 cases of healthy control (healthy control group) were selected from August 2016 to May 2017. The gene expression was detected in 4 ml peripheral anticoagulant blood by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the relative expression of two genes in three groups were compared. Results The GBP5 gene expression in three groups was significantly differentce (F=7.23, P=0.001). The GBP5 gene relative expression in pulmonary tuberculosis group was significantly higher than that in latent infection group :1.58 ± 0.80 vs. 1.09 ± 0.68, there was significant difference (t=2.93, P=0.004). The GBP5 gene relative expression in pulmonary tuberculosis group was significantly higher than that in healthy control group: 1.58 ± 0.80 vs. 1.04 ± 0.61, there was significant difference (t=3.40, P=0.010). The GBP5 gene relative expression in latent infection group and healthy control group had no significant difference (t=0.39, P=0.700). There was no significant difference in ASAP1 expression among three groups (F=0.26, P=0.770). Conclusions The expression of GBP5 in pulmonary tuberculosis patients, latent tuberculosis infection patients and healthy controls is different, and GBP5 could screen latent tuberculosis infection patients which is expected to be a potential screening marker for latent tuberculosis infection.

Expresssion of miRNA-1 in esophageal squamous cell carcinoma tissues and its mechanism / 肿瘤

Objective: To investigate the expression of microRNA-1 (miRNA-1) in human esophageal squamous cell carcinoma (ESCC) tissues and its possible mechanism. Methods: The expression levels of miRNA-1 and LIM and SH3 domain protein 1 (LASP1) mRNA and LASP1 protein in 55 cases of ESCC and its adjacent para-cancerous tissues were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The potential target gene of miRNA-1 was predicted by online bioinformatics software. The target gene of miRNA-1 after Eca109 cells co-transfection with recombination dual luciferase reporter vector psiCHECK-2-LASP1 containing 3'-untranslated region (3'-UTR) with miRNA-1 binding site of LASP1 gene and miRNA-1 mimic was verified by dual-luciferase reporter assay system. The expressions of miRNA-1 and LASP1 protein in Eca109 cells after transfection with miRNA-1 mimic or miRNA-1 inhibitor were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. Results: The expression level of miRNA-1 in ESCC tissues was lower than that in adjacent para-cancerous tissues (P < 0.01), and the expression levels of LASP1 mRNA and protein were opposite (all P < 0.05). The expressions of miRNA-1 and LASP1 were associated with lymph metastasis, TNM staging and histological grade (all P < 0.05). The miRNA-1 expression in ESCC tissues was positively associated with LASP1 (r = +0.45, P < 0.05). LASP1 was target gene of miRNA-1. miRNA-1 could directly target the LASP1 3'-UTR. The expression level of miRNA-1 was up-regulated in Eca109 cells after transfection with miRNA-1 mimic (P < 0.01), and the expression level of LASP1 protein was down-regulated (P < 0.05). The expression level of miRNA-1 was down-regulated in Eca109 cells after transfection with miRNA-1 inhibitor (P < 0.05), and the expression level of LASP1 protein was up-regulated (P < 0.05). Conclusion: The expression level of miRNA-1 is low in ESCC tissues, and its mechanism may be associated with regulation of target gene LASP1.

Lack of association between polymorphisms in the UBASH3A gene and autoimmune thyroid disease: a case control study / Falta de associação entre polimorfismos do gene UBASH3A e doença tiroidiana autoimune: estudo caso controle

Objective: The aim of this study was to investigate UBASH3A gene variation association with autoimmune thyroid disease and clinical features in a Chinese Han population. Subjects and methods: A total of 667 AITD patients (417 GD and 250 HT) and 301 healthy controls were genotyped for two single nucleotide polymorphisms (SNPs) rs11203203, rs3788013 of UBASH3A gene, utilizing the Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) Platform. Results: Between the control group and AITD, GD and HT group, no statistically significant difference was observed in the genotypic and allelic frequencies of the two SNPs. There was no significant difference in allelic frequencies of the two SNPs between GD with and without ophthalmopathy. There was no significant difference in haplotype distributions between the control group and AITD, GD or HT group. Conclusion: Rs11203203 and rs3788013 in UBASH3A gene may not be associated with AITD patients in Chinese Han population. .

Objetivo: O objetivo deste estudo foi investigar a variação no gene UBASH3A com a doença tiroidiana autoimune e características clínicas na população chinesa Han. Sujeitos e métodos: Um total de 667 pacientes com DTAI (417 com DG e 250 com TH) e 301 controles saudáveis foi genotipado para dois polimorfismos de nucleotídeo simples (SNPs) rs11203203, rs3788013 do gene UBASH3A, usando-se a plataforma MALDI-TOF-MS (Ionização/Dessorção de Matriz Assistida por Laser – Tempo de Voo/Espectrômetro de Massa). Resultados: Não foram observadas diferenças significativas entre as frequências genotípicas e alélicas dos dois SNPs nos grupos controle e DTAI, DG e TH. Não houve diferenças significativas entre as frequências alélicas dos dois SNPs em pacientes com DG com ou sem olftalmopatia. Não houve diferenças significativas nas distribuições de haplótipos no grupo controle e nos grupos DTAI, DG e TH. Conclusão: Os SNPs rs11203203 e rs3788013 do gene UBASH3A podem não estar associados a pacientes com DTAI na população chinesa Han. .

Aggregation properties of a short peptide that mediates amyloid fibril formation in model proteins unrelated to disease.

Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The hexapeptide stretch 21DIDLHL26 has been shown to be important in the self-assembly of the Src homology 3 (SH3) domain of p85α subunit of bovine phosphatidylinositol-3-kinase (PI3-SH3). The SH3 domain of chicken brain α- spectrin, which is otherwise non-amyloidogenic, is rendered amyloidogenic if 22EVTMKK27 is replaced by DIDLHL. In this article, we describe the aggregation behaviour of DIDLHL-COOH and DIDLHL-CONH2. Our results indicate that DIDLHL-COOH and DIDLHL-CONH2 aggregate to form spherical structures at pH 5 and 6. At pH 5, in the presence of mica, DIDLHL-CONH2 forms short fibrous structures. The presence of NaCl along with mica results in fibrillar structures. At pH 6, DIDLHL-CONH2 forms largely spherical aggregates. Both the peptides are unstructured in solution but adopt β-conformation on drying. The aggregates formed by DIDLHL-COOH and DIDLHL-CONH2 are formed during drying process and their structures are modulated by the presence of mica and salt. Our study suggests that a peptide need not have intrinsic amyloidogenic propensity to facilitate the selfassembly of the full-length protein. The propensity of peptides to form self-assembled structures that are nonamyloidogenic could be important in potentiating the self-assembly of full-length proteins into amyloid fibrils.

The influence of a Grb2 inhibitor on K562 cell growth / 肿瘤研究与临床

Objective To analyze the effects of an inhibitor of the SH3 (Src homology) domains of Grb2 on the growth and proliferation of K562 cells. Methods The peptidimer [(VPPPVPPRRR)2-K], penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c [poptidimer linked to penetratin: (VPPPVPPRRR)2-K-Aha-RQIKIWFQNRRMKWKK] were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy (MS). A pull-down assay was used to observe the specific binding of peptidimer-c to the Grb2 of K562 cell lysates. The inhibition of peptidimer-c on K562 cell proliferation was evaluated by trypan blue exclusion assay, the cytostatic effect was tested by clonogenic assay, and the cytotoxicity was examined by WST-1 method. A further experiment was performed with clonogenic assay to analyze the co-effect of peptidimer-c respectively combined with Gleevec, Hydroxyurea and Cytarabine by Jing's method. Results The HPLC analysis showed only a simple peak, which means that the peptide is in high purity. MS analysis showed the peptides were coincided with the design. The molecular weight of peptidimer-c was of 4794.0 and that of the penetratin 2246.7. Pull-down assay demonstrated that the peptidimer-c, not the penetratin, could bind to Grb2 specifically. The trypan blue assay showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, even 3～6 h after the cells were exposed to the drug, and penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-1 test showed the cytotoxieity of peptidimer-c or Gleevec on K562 cells, the IC50 of peptidimer-c was (17±2) μmol/L and the IC50 of Gleevec was (0.25±0.05) μmol/L. In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. The IC50 value ofpeptidimer-c on K562 colony formation was (3.9±0.9) μmol/L, IC50 of Gleevec was (0.03±0.02) μmol/L, IC50 of Hydroxyurea was (15±7) μmol/L, and that of cytarabine was (0.014±0.012) μmol/L. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5 μmol/L peptidimer-c and 0.05 μmol/L Gleevec showed synergistic effect on K562, as well as the combination of 1.5 μmol/L peptidimer-c and 0.006 μmol/L or 0.01 μmol/L Cytarabine. Conclusion These results suggested that peptidimer-c had an inhibitory effect on K562 cells and combination of peptidimer-c with other drugs would increase the anti-cancer effects.

Distinct Regional and Cellular Localization of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 1 in Cerebellar Cortex of Rat

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarizationactivated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. METHODS: HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using softwaresearching method. RESULTS: HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. CONCLUSION: Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.

Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

A Study on the Proteins that Interact with Human Nebulin SH3 Domain / 대한정형외과연구학회지

OBJECTIVE: bjective: By identifying the unknown substance responsible for binding with nebulin SH3 domain within the sarcomeric Z-line, we tried to find out Z-line structure which plays an important role on muscle contraction and maintenance of muscle funtion. METHOD: First, the bait plasmid was made by binding the DNA binding domain of Gal4 protein of yeast and the SH3 domain. Second, library plasmid was made by binding activation domain and human skeletal cDNA library. Then, the base sequence of the clone, produced by combining the two proteins expressed by transgenically converted plasmid in yeast, was analyzed. RESULT: We screened out six true positive clones and analyzed the base sequence of the two of six clones. We identified them to be alpha-actinin2. CONCLUSION: We can theorize that Neublin SH3 domain and alpha-actinin2 plays a vital role for the integration of Z-line. Thus, this is an important data in further studying muscle functions, mechanisms, and muscular disease as well.

Cell-free expression and functional reconstitution of CALM in clathrin assembly

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.

Properties of GST-CALM expressed in E. coli

Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.