OBJECTIVETo investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells.

METHODSA synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed.

RESULTSCompared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01).

CONCLUSIONAnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.

Annexin A2 ; genetics ; metabolism ; Cell Proliferation ; Chemotaxis ; genetics ; Gene Silencing ; Humans ; Jurkat Cells ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVE: To investigate the enhancement of macrophage chemotaxis in patients with knee osteoarthritis (KOA) and its correlation with the disease severity. METHODS: Eighty patients with KOA admitted from July 2019 to June 2022 were enrolled as the observation group and divided into 29 cases of moderate group, 30 cases of severe group and 21 cases of extremely severe group. At the same time, 30 healthy subjects were included as the control group. The gene expressions of NF-κB, CXC chemokine receptor 7 (CXCR7) and CXC chemokine ligand 12 (CXCL12) in macrophages of each group were analyzed. Visual analogue scale(VAS) was used to evaluate the degree of joint pain. Joint function was evaluated by knee Joint Society Scoring system(KSS). Finally, data analysis was carried out. RESULTS: The expression levels of NF-κB, CXCR7 and CXCL12 in moderate group, severe group and extreme recombination group were higher than those in control group. The VAS, the expression of NF-κB, CXCR7 and CXCL12 in the severe group and the extreme recombination group were higher than those in the moderate group, whereas KSS was lower than that in the moderate group. The VAS, expression levels of NF-κB, CXCR7 and CXCL12 in the extremely severe group were higher than those in the severe group, and KSS was lower than that in the severe group (all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with VAS score, but negatively correlated with KSS(all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with the severity of disease. After excluding the influence of traditional factors (gender, age and disease duration), multiple linear regression analysis further showed that the expression levels of NF-κB, CXCR7 and CXCL12 were still positively correlated with the severity of disease(all P<0.01). CONCLUSION The chemotaxis of macrophages in patients with KOA increased with the aggravation of the disease, and was related to the degree of pain and function impairment.
Humans ; Osteoarthritis, Knee/genetics* ; Chemotaxis/genetics* ; NF-kappa B/metabolism* ; Macrophages/metabolism* ; Receptors, CXCR/metabolism* ; Patient Acuity

OBJECTIVETo construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization.

METHODSThe plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.

RESULTSThe fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05).

CONCLUSIONThe function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Animals ; Bacterial Proteins ; genetics ; Campylobacter jejuni ; genetics ; pathogenicity ; Chemotaxis ; genetics ; Gene Knockout Techniques ; Jejunum ; microbiology ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C

Animals integrate various environmental stimuli within the nervous system to generate proper behavioral responses. However, the underlying neural circuits and molecular mechanisms are largely unknown. The insulin-like signaling pathway is known to regulate dauer formation, fat metabolism, and longevity in Caenorhabditis elegans (C. Elegans). Here, we show that this highly conserved signaling pathway also functions in the integrative response to an olfactory diacetyl and a gustatory Cu(2+) stimuli. Worms of wild-type N2 Bristol displayed a strong avoidance to the Cu(2+) barrier in the migration pathway to the attractive diacetyl. Mutants of daf-2 (insulin receptor), daf-18 (PTEN lipid phosphatase), pdk-1 (phosphoinositide-dependent kinase), akt-1/-2 (Akt/PKB kinase) and sgk-1 (serum- and glucocorticoid-inducible kinase) show severe defects in the elusion from the Cu(2+). Mutations in DAF-16, a forkhead-type transcriptional factor, suppress the integrative defects of daf-2 and akt-1/-2 mutants. We further report that neither cGMP nor TGFβ pathways, two other dauer formation regulators, likely plays a role in the integrative learning. These results suggest that the insulin-like signaling pathway constitutes an essential component for sensory integration and decision-making behavior plasticity.
Animals ; Caenorhabditis elegans ; genetics ; physiology ; Caenorhabditis elegans Proteins ; genetics ; physiology ; Chemotaxis ; genetics ; physiology ; Copper ; physiology ; Cyclic GMP ; genetics ; physiology ; Diacetyl ; metabolism ; Insulin ; metabolism ; Longevity ; Signal Transduction ; Smell ; genetics ; physiology ; Taste ; genetics ; physiology ; Transforming Growth Factor beta ; genetics ; physiology

OBJECTIVETo clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods.

METHODSWith the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method.

RESULTSA 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes.

CONCLUSIONThe prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.

Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Computational Biology ; methods ; Genetic Vectors ; genetics ; Helicobacter hepaticus ; genetics ; isolation & purification ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Methyl-Accepting Chemotaxis Proteins ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo suppress the expression of CCR5 and CXCR4, the co-receptors for human immunodeficiency virus type 1 ( HIV-1), and thus inhibit HIV-1 from entering cells.

METHODSDNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells (PBMCs) by reverse transcript polymerase chain reaction (RT-PCR) and sequencing was performed. Correct fragments were inserted into Shuttle plasmid inversely, which was recombined with backbone plasmid containing homologous adenoviral genome in E. coli BJ5183. The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified. Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR, and the titres of them were determined by cytopathic effect (CPE) method. The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5 (multiplicity of infection, MOI = 100) and CXCR4 (MOI = 200), respectively. The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24, 48, 72 hours and 10 days after infection. In addition, the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively.

RESULTSWe constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively. The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5 x 10" PFU/ml and 7 x 10(10) PFU/ml, respectively. The expression rate of CCR5 on U937 cells decreased from 82. 10% (blank control) to 1.12% (Ad-antiR5 infected) , and that of CXCR4 on MT4 cells decreased from 42% (blank control) to 1.03% (Ad-antiX4 infected) 24 hours later. The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02% , 1.26% , 1.23% at 48 hours, 72 hours, and 10 days later, respectively. The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%, 1.17%, 1.22% at 48 hours, 72 hours, and 10 days later, respectively. Moreover, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells.

CONCLUSIONThe recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.

Adenoviridae ; genetics ; Cell Line, Transformed ; Cell Proliferation ; Chemotaxis ; Down-Regulation ; Genetic Vectors ; Humans ; RNA, Antisense ; genetics ; RNA, Messenger ; genetics ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, CXCR4 ; biosynthesis ; genetics ; Transfection ; U937 Cells

BACKGROUNDCC chemokine receptor 3 (CCR3), expressed on some inflammatory cells, is a member of the chemokine receptor family. Its ligand is eotaxin/CCL11. In this research, we studied the expression and function of CCR3 induced by interleukin-2 (IL-2) and interleukin-4 (IL-4) on human germinal centre (GC) B cells.

METHODSCells isolated from human tonsils were stimulated with IL-2 or/and IL-4 followed by bonding with eotaxin/CCL11. Flow cytometry was used to detect expression of CCR3 on GC B cells and apoptosis of GC B cells. Real time quantitative reverse transcription polymerase chain reaction and Northern blot assays were used to analyse the CCR3 mRNA expressed in the GC B cells. Chemotaxis and adhesion assays were used to determine the effect of eotaxin/CCL11 ligand bonded to CCR3 on GC B cells.

RESULTSThere was no CCR3 expression on human freshly isolated GC B cells. The combination IL-2 and IL-4 could upregulate CCR3 mRNA and protein expression on GC B cells. Eotaxin could not induce GC B cell chemotaxis and adhesion but triggered apoptosis of GC B cells.

CONCLUSIONIL-2 and IL-4 together induced expression of CCR3 on GC B cells, and the receptor acted as a death receptor.

Apoptosis ; B-Lymphocytes ; metabolism ; pathology ; Cell Adhesion ; Chemotaxis, Leukocyte ; Germinal Center ; metabolism ; pathology ; Humans ; Interleukin-2 ; pharmacology ; Interleukin-4 ; pharmacology ; RNA, Messenger ; analysis ; Receptors, CCR3 ; Receptors, Chemokine ; genetics

OBJECTIVETo investigate the role of CD147 in the proliferation, activation and chemotaxis of Jurkat cell induced by cyclophilin A (CyPA).

METHODSCD147 mRNA and protein level siRNA transfected Jurkat cells were identified by RT-PCR and Western blot respectively. Jurkat cell, Jurkat-vector cell and Jurkat-CD147 siRNA cells were treated with different concentrations of CyPA (0.01 nmol/L, 0.1 nmol/L, 1 nmol/L, 10 nmol/L) or PBS for 24 h and 48 h. Proliferation level was detected by MTT assay. CD25 was measured by flow cytometry. Transwell chamber was used to detect the chemotaxis. The effect of CyPA on the adhesive potential of Jurkat cell was studied by cell-matrix adhesion assay.

RESULTSCD147 mRNA and protein level siRNA transfected cells were decreased significantly than that of control cells. CyPA stimulated the proliferation of Jurkat cell in a dose-dependent manner, its effect peaked at 10 nmol/L CyPA. Blockage of CD147 expression decreased the proliferation level of Jurkat cell induced by CyPA. CyPA increased the activation rate of Jurkat cell, and blockage CD147 expression decreased the activation rate of the cell induced by CypA. CyPA showed a chemotactic activity on Jurkat cell, the chemotaxis index being 2.32, and the chemotactic ability was decreased after inhibition of CD147 expression. CyPA had no effect on adhesion of Jurkat cell to extracellular matrix.

CONCLUSIONCD147 plays a role in the proliferation, activation and chemotaxis of Jurkat cell induced by CyPA.

Basigin ; genetics ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Chemotaxis ; drug effects ; Cyclophilin A ; pharmacology ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Jurkat Cells ; RNA, Small Interfering ; genetics ; Transfection

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemotaxis, Leukocyte/physiology ; Diamide/*pharmacology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Lipid Peroxidation ; Macrophage Inflammatory Protein-1/*biosynthesis ; Macrophage Inflammatory Protein-1/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sulfhydryl Reagents/pharmacology ; Umbilical Veins/cytology

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; physiology ; Diamide ; pharmacology ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipid Peroxidation ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Sulfhydryl Reagents ; pharmacology ; Umbilical Veins ; cytology