Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

AIM： To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy. METHODS： Nuclear matrix proteins were selectively extracted from MGcS0-3 cells treated with or without hexamethylamine bisacetamide （HMBA）, and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ ionization time of flight mass spectrometry （MALDI-TOFMS） analysis and submitted for database searching using Mascot tool. RESULTS： The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGcS0-3 cells compared to control. Eleven of which were identified. Seven proteinsactin, prohibitin, porin 31HL, heterogeneous nuclear dbonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells. CONCLUSION： The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

STUDY ON NUCLEAR MATRIX PROTEINS FROM HUMAN BREAST CARCINOMA

Objective To investigate the marker protein of human breast carcinoma from nuclear matrix proteins (NMPs). Methods NMPs were injected subcutaneously into rabbit to get antiserum, which was used to detect the NMPs specificity for breast carcinoma. Results There was an apparent positive band (100 kD) in the NMPs of breast carcinoma, which did not exist in normal breast and other tumors that were detected.Conclusion One or one group of 100 kD NMPs were found to be related to human breast carcinoma, which may be involved in the carcinogenesis and development of human breast carcinoma and valuable for breast carcinoma diagnosis.

Alreration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM： To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS： Cells cultured with or without 5 × 10^-3 mmol/L of hexamethylene bisacetamide （HMBA） on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser- desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Data were submitted for database searching using Mascot tool （www.matrixscience.com）. RESULTS： The nuclear matrix （NM） and intermediate filament （IF） in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate- sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pystl, hypothetical protein, nucleophosminl, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, 13-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION： The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament （NM-IF） system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.

Interactions between the nuclear matrix proteins and the 5'-flanking cis-acting elements of the human ε-globin gene

An erythroid-specific nuclear matrix protein (termed ε-NMP_k) in K562 cells, which can specifically bind to the positive stage-specific regulatory element (ε-PRE Ⅱ, -446—-419 bp) upstream of the human ε-globin gene, has been identified by using gel mobility shift assay.Meanwhile, Southwestern blotting assay showed that the nuclear matrix protein ε-NMP_k in K562,cells may be composed of two polypeptides ( ～ 40 ku). In addition, it is observed in the gel mobility shift assay that the nuclear matrix proteins from K562, HEL and Raji cells can bind to the silencer DNA ( - 392— -177 bp) in the 5’-flanking sequence of human ε-globin gene respectively. However, the shift band K detected in K562 cells is different from shift band H/R in HEL and Raji cells, suggesting that a common nuclear matrix protein may exist in HEL and Raji cells. Results show that the nuclear matrix protein may play an important role in the regulation of the human ε-globin gene expression.

The interaction between the human β-globin locus control region and nuclear matrix

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express humanβ-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681- -10971 bp , HS3 core sequence -14991- -14716 bp and HS4 core sequence -18586- -18306 bp) of DNase I hypersensitive sites in the human β-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of humanβ-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of β-globin gene and to promote its transcription.

NUCLEAR MATRIX PROTEIN IN LEUKEMIA CELLS

Objective: To compare the composition of nuclear matrix proteins (NMP) between leukemia cells and normal bone marrow cells. Methods: NMP was isolated by high-salt extraction and identified in acute and chronic myelogenous leukemia cells as well as in the blast phase of chronic leukemia. On SDS-PAGE, NMPs with molecular myelogenous ferment from what were seen in normal bone marrow cells were present in both acute and chronic myelogenous leukemia. Conclusion: Marked changes of NMP, not only in contents but also in compositions, exist in leukemic cells compared with normal bone marrow cells. NMP may serve as a target of chemotherapeutic drug against leukemia.

Comparisons of voided urine cytology, nuclear matrix protein-22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China

Aim： To compare the results of bladder tumor associated antigen （BTA TRAK）, nuclear matrix protein 22 （NMP 22） and voided urine cytology （VUC） in detecting bladder cancer. Methods： A total of 135 elderly male and 50 healthy volunteers enrolled in this study were classified into three groups： （i） 93 patients with bladder cancer; （ii） 42 patients with urinary benign conditions; and （iii） 50 healthy volunteers. BTA TRAK and NMP 22 kits were used to detect bladder cancer. Voided urine cytology was used to compare the sensitivity and specificity of the screening tests. Results： The sensitivity and specificity of cytology, BTA TRAK and NMP 22 were 24% and 97%, 51% and 73%, 78% and 73%, respectively. The level of NMP 22 increased with tumor grading. The BTA TRAK kit has the lowest sensitivity among the screening tests. The NMP 22 with the best sensitivity can be an adjunct to cytology for evaluating bladder cancer. Conclusion： The NMP 22 test has a better correlation with the grading of the bladder cancer than BTA TRAK. As cytology units are typically not available in hospitals or in outpatient clinics, NMP 22 might be a promising tool for screening bladder cancer.

CHANGES OF NUCLEAR MATRIX PROTEIN AND ITS RELATIONSHIP WITH c-erbB-2 IN HUMAN COLON ADENOCARCINOMA

Objective： Nuclear matrix protein is tissue, cell-type specific, and tumor-relative. It plays an important role in the regulation of intranuclear processes. Some researches also showed that a c-erbB-2 promoter-specific DNA-binding nuclear matrix protein is present only in malignant human breast tissues and induces mitogenesis and cell surface expression of the c-erbB-2 protein in resting NIH/3T3 cells. But it is not clear that how it in colon adenocarcinomas. Methods： Two-dimensional gel electrophoretic method was used for NMP identification and immunohistochemistry was used for c-erbB-2 detection in 12 cases of colon adenocarcinomas and matched adjacent normal colon tissues. Results： 5 different nuclear matrix proteins （named C1-C5） were identified in 12 colon adenocarcinoma specimens, but not in the matched adjacent normal colon tissues; 3 nuclear matrix proteins （named N1-N3） were identified in all 12 matched adjacent normal colon tissues, but not in colon adenocarcinoma specimens. A nuclear matrix protein （named N4） was detected in all of 9 moderated-well differentiated adenocarcinomas and all 12 matched adjacent normal colon tissues, but not in 3 poor-differentiated adenocarcinomas. All of the 10 colon adenocarcinomas which had the nuclear matrix protein C4 were c-erbB-2 expression positive. Conclusion： The data suggest that there are specific nuclear matrix proteins in colon adenocarcinomas and its subtypes, which maybe valuable to serve as markers of colon adenocarcinomas in future. Nuclear matrix protein C4 probably is a c-erbB-2 promotor-specific nuclear matrix protein in colon adenocarcinomas, and may induce the expression of c-erbB-2.

抗NXP2抗体阳性的皮肌炎1例报告并文献复习

抗核基质蛋白2(nuclear matrix protein 2,NXP2)抗体阳性的皮肌炎是特发性炎性肌病的一种,除肌无力、皮肤改变的典型表现外,还有钙质沉着、皮下水肿、严重的肌痛及吞咽困难等表现。本文报道1例具有典型临床及影像学表现、随访17个月后复查抗NXP2抗体为阴性的患者。1病例资料患者,男,27岁,因“肢体疼痛无力1月余,加重2周”于2019年6月8日就诊于我科。患者于入院1月前无明显诱因出现肢体疼痛,腰部疼痛症状明显。3周前出现肢体无力感,伴面部皮疹,鼻翼两侧为著。2周前出现全身浮肿,抬头及转颈无力,双上肢抬起费力,坐起费力,行走约50米后肌肉无力酸痛明显,伴双上肢近端、双侧髋部、双膝关节及前胸部皮疹,伴发热,化验示肌酸激酶7500 U/L,行肌电图示肌源性损害,门诊按“肌病”收至我科。患者自发病以来,饮食睡眠可,二便如常,体重增加约10千克。

Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury

Propofol has been shown to exert neuroprotective effects on the injured spinal cord.However,the effect of propofol on the blood-spinal cord barrier（BSCB） after ischemia/reperfusion injury（IRI） is poorly understood.Therefore,we investigated whether propofol could maintain the integrity of the BSCB.Spinal cord IRI（SCIRI） was induced in rabbits by infrarenal aortic occlusion for 30 minutes.Propofol,30 mg/kg,was intravenously infused 10 minutes before aortic clamping as well as at the onset of reperfusion.Then,48 hours later,we performed histological and m RNA/protein analyses of the spinal cord.Propofol decreased histological damage to the spinal cord,attenuated the reduction in BSCB permeability,downregulated the m RNA and protein expression levels of matrix metalloprotease-9（MMP-9） and nuclear factor-κB（NF-κB）,and upregulated the protein expression levels of occludin and claudin-5.Our findings suggest that propofol helps maintain BSCB integrity after SCIRI by reducing MMP-9 expression,by inhibiting the NF-κB signaling pathway,and by maintaining expression of tight junction proteins.

Biomimetic synthesis of all-inclusive organic-inorganic nanospheres for enhanced electrochemical immunoassay

A friendly biomimetic process was adopted for the mild preparation of"all-inclusive"organic-inorganic nanospheres,which effectively integrate biorecognition function and signal amplification function.The resulted Ca3(PO4)2-Ab2-BSA nanospheres were employed as signal labels for enhancing detection of nuclear matrix protein 22(NMP 22).The fabricated electrochemical immunosensor exhibited a linear range(0.08-77.00 U/mL)and an ultralow limit of detection(0.01 U/mL)towards NMP 22,which can be taken as a promising tool for clinical diagnosis of bladder cancer.