OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

Gap prepulse inhibition of acoustic startle (GPIAS) method has been used effectively for the objective assessment of tinnitus in animals. Among two types of enclosures for the GPIAS, the unconstrained type carries less risk of animal death due to the absence of binding stress in the enclosure, and lack of need for alteration to animal size variation as it grows. However, animals' voluntary movements, which have no relation to the startles evoked by acoustic stimuli, are problematic, as they cannot be excluded in the case of the unconstrained enclosure based GPIAS measurement system. In order to discount voluntary movements which are not associated with external acoustic stimuli, we propose the conditional random interstimulus interval (CR ISI) method for unconstrained enclosure based GPIAS measurement. With the proposed ISI method, the unconstrained enclosure based acoustic startle response measurement system has been implemented in this paper. As a result, the effectiveness of the proposed CR ISI method has been verified and compared with those of conventional ISI methods through animal experiments using SD-rats. The experimental results showed that abnormal startle responses and invalid GPIAS values caused by motion were prevented when our proposed CR ISI method was applied to our implemented system. It was also verified that our proposed CR ISI method is advantageous in reducing the total experimental time for acquiring normal startle responses and valid GPIAS values, compared to conventional ISI methods, since our proposed CR ISI can begin the acoustic stimulation only when the animal gets stable and motionless.
Acoustic Stimulation ; Acoustics ; Animal Experimentation ; Animals ; Integrin alpha2 ; Methods ; Prepulse Inhibition ; Reflex, Startle ; Tinnitus

Memantine, a noncompetitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, suppresses the release of excessive levels of glutamate that may induce neuronal excitation. Here we investigated the effects of memantine on salicylate-induced tinnitus model. The expressions of the activity-regulated cytoskeleton-associated protein (ARC) and tumor necrosis factor-alpha (TNF α)genes; as well as the NMDA receptor subunit 2B (NR2B) gene and protein, were examined in the SH-SY5Y cells and the animal model. We also used gap-prepulse inhibition of the acoustic startle reflex (GPIAS) and noise burst prepulse inhibition of acoustic startle, and the auditory brainstem level (electrophysiological recordings of auditory brainstem responses, ABR) and NR2B expression level in the auditory cortex to evaluate whether memantine could reduce salicylate-mediated behavioral disturbances. NR2B was significantly upregulated in salicylate-treated cells, but downregulated after memantine treatment. Similarly, expression of the inflammatory cytokine genes TNFα and immediate-early gene ARC was significantly increased in the salicylate-treated cells, and decreased when the cells were treated with memantine. These results were confirmed by NR2B immunocytochemistry. GPIAS was attenuated to a significantly lesser extent in rats treated with a combination of salicylate and memantine than in those treated with salicylate only. The mean ABR threshold in both groups was not significant different before and 1 day after the end of treatment. Additionally, NR2B protein expression in the auditory cortex was markedly increased in the salicylate-treated group, whereas it was reduced in the memantine-treated group. These results indicate that memantine is useful for the treatment of salicylate-induced tinnitus.
Acoustics ; Animals ; Auditory Cortex ; Brain Stem ; Evoked Potentials, Auditory, Brain Stem ; Genes, Immediate-Early ; Glutamic Acid ; Immunohistochemistry ; Integrin alpha2 ; Memantine ; Models, Animal ; N-Methylaspartate ; Neurons ; Noise ; Prepulse Inhibition ; Rats ; Reflex, Startle ; Tinnitus ; Tumor Necrosis Factor-alpha

UNLABELLEDOBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.

METHODSStable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3ΔNITY (β3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and β3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions.

RESULTSCHO-αIIbβ3, CHO-αIIbβ3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbβ3 had the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbβ3. The β3ΔNITY mutation substantially reduced kindlin-2 association.

CONCLUSIONDeletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin β3, thereby partially inhibit the integrin β3 signaling.

Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Fibrinogen ; Humans ; Integrin alpha2 ; Integrin beta3 ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Structure, Tertiary ; Signal Transduction

integrin alpha2 (alpha2) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the selective cell death effect of anti-integrin alpha2 and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticles (GNP) with air plasma for selective cell death of oral SCC.METHODS: Expression of integrin alpha2, EGFR on human SCC cells (SCC25) were examined by western blot. SCC25 cells were treated with anti-integrin alpha2, anti-EGFR and analysed by Hemacolor staining, immunoflorescence staining, FACS flow cytometry. Conjugated GNP with integrin alpha2, EGFR antibody were treated by air plasma on SCC cells.RESULTS: Integrin alpha2 and EGFR were over-expressed on SCC25 cells than normal lung WI-38 cells. The cell viability rate of SCC25 cells treated with anti-integrin alpha2, anti-EGFR was lower than WI-38 cells. The concentration changes of nucleus, releasing cytochrome c and apoptosis inducing factor (AIF) from mitochondria to cytosol were observed. The changes of proteins related with apoptosis were observed. Increase of bax, bcl-xL, activation of caspase-3, -7, -9, and fragmentation of PARP, DFF45 and decrease of lamin A/C in SCC25 cells were observed. In FACS, increase of sub-G1 and S phase was observed. Cell cycle related proteins, Such as cyclin D1, cyclin dependent kinase (CDK) 4, cyclin A, cyclin E, CDK 2, p27 were decreased. After SCC25 cells treated with conjugatged GNP-Integrin alpha2, GNP-EGFR, additionally air plasma, the cell death rate was significantly increased.CONCLUSION: Integrin alpha2, EGFR were over-expressed in oral SCC cells. Anti-integrin alpha2, anti-EGFR in SCC25 cells induced apoptosis selectively. When GNP-anti integrin alpha2, GNP-anti EGFR were treated with air plasma on SCC25 cells, cancer cells were died more selectively. GNP-anti integrin alpha2, GNP-anti EGFR with air plasma could be treatment choice of oral SCC.]]>
Apoptosis ; Apoptosis Inducing Factor ; Blotting, Western ; Carcinoma, Squamous Cell ; Caspase 3 ; Cell Cycle ; Cell Death ; Cell Survival ; Cyclin A ; Cyclin D1 ; Cyclin E ; Cyclins ; Cytochromes c ; Cytosol ; Flow Cytometry ; Humans ; Integrin alpha2 ; Lung ; Mitochondria ; Nanoparticles ; Phosphotransferases ; Plasma ; Proteins ; Receptor, Epidermal Growth Factor ; S Phase

BACKGROUND/AIMS: For unknown reasons, caspase-1 -/- mice, protected against cisplatin-induced acute renal failure (ARF), are deficient in interleukin (IL)-1alpha. We thus asked whether IL-1alpha deficiency underlies the mechanism of protection against cisplatin-induced ARF in these mice. METHODS: Cisplatin (30 mg/kg) was injected intraperitoneally into wild-type C57BL/6 mice to produce a cisplatin-induced model of ARF. IL-1alpha was measured in control vehicle- and cisplatin-treated wild-type animals. We also examined whether IL-1alpha -/- mice were similarly protected against cisplatin-induced ARF. Additionally, infiltration of CD11b- and CD49b-positive cells, as markers of macrophages, natural killer, and natural killer T cells (pan-NK cells), was investigated in wild-type and IL-1alpha -/- mice. RESULTS: Compared with vehicle-treated mice, renal IL-1alpha increased in cisplatin-treated wild-type mice beginning on day 1. IL-1alpha -/- mice were shown to be protected against cisplatin-induced ARF. No significant difference in the infiltration of neutrophils or CD11b- and CD49b-positive cells were observed between wild-type and IL-1alpha -/- mice. CONCLUSIONS: Mice deficient in IL-1alpha are protected against cisplatin-induced ARF. The lack of IL-1alpha may explain, at least in part, the protection against cisplatin-induced ARF observed in caspase-1 -/- mice. Investigation of the protective mechanism (s) in IL-1alpha -/- mice in cisplatin-induced ARF merits further study.
Acute Kidney Injury/chemically induced/*immunology/pathology/physiopathology/prevention & control ; Animals ; Antigens, CD11b/analysis ; Apoptosis ; Biological Markers/blood ; Blood Urea Nitrogen ; *Cisplatin ; Creatinine/blood ; Disease Models, Animal ; Fluorescent Antibody Technique ; Integrin alpha2/analysis ; Interleukin-1alpha/deficiency/genetics/*metabolism ; Kidney/*immunology/pathology/physiopathology ; Killer Cells, Natural/immunology ; Macrophages/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Natural Killer T-Cells/immunology ; Necrosis ; Neutrophil Infiltration ; Time Factors

BACKGROUNDGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China.

METHODSA three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping.

RESULTSThe proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately.

CONCLUSIONSThe alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

Asian Continental Ancestry Group ; Child ; Female ; Flow Cytometry ; Heterozygote ; Humans ; Integrin alpha2 ; genetics ; Integrin beta3 ; genetics ; Mutation ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction ; Thrombasthenia ; genetics ; metabolism ; pathology

OBJECTIVETo observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action.

METHODSAttachment assay was used to assess the adhesion of wwox-transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells. Integrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane.

RESULTSAttachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours (P<0.01). The level of membranous integrins alpha2 and alpha3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in vector-transfected controls (P<0.05), but there was no association with the level of functioning integrins betal or beta2 in clone cells (P>0.05). The attachment assays were repeated after pre-incubating the cells with integrin alpha2 or alpha3 function-blocking antibodies. These results showed that blocking integrin alpha3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody (P<0.05). In contrast, preincubation with alpha2 blocking antibody had very little effect on fibronectin binding in these cells (P>0.05). FACS analysis showed that membranous integrin alpha3 expression revealed a marked reduction in WWOX-transfected cells than that in vector-transfected cells.

CONCLUSIONWWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.

CD18 Antigens ; metabolism ; Cell Adhesion ; Female ; Fibronectins ; metabolism ; Humans ; Integrin alpha2 ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Oxidoreductases ; genetics ; metabolism ; Protein Binding ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo study the association of integrin alpha-2 (ITGA2) gene C807T, integrin beta-3 (ITGB3) gene T176C polymorphisms with ischemic stroke and the effect of the polymorphisms on plasma lipid and lipoprotein levels.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to detect the integrin genotypes in 265 patients with ischemic stroke and 280 healthy controls. The plasma lipid and lipoprotein levels were measured by routine method.

RESULTSPlasma total cholesterol (TC), triacylglycerol (TG) and low density lipoprotein-cholesterol (LDL-C) in the patients with ischemic stroke were significantly higher than those in the controls (P< 0.05). The distributions of the ITGB3 gene T176C polymorphism were not different between the ischemic stroke group and control group, but the ITGA2 gene C807T polymorphism was significantly different. The relative risk suffering from ischemic stroke of the T allele carrier was 1.455 times as that of the C allele carrier (OR=1.455, 95%CI: 1.134-1.866). The level of plasma lipid in the T allele carriers was significantly higher than that in the C allele carriers (P< 0.05).

CONCLUSIONThe ITGA2 gene C807T polymorphism was associated with ischemic stroke, the 807 T allele may be a genetic risk factor for ischemic stroke. The ITGA2 gene C807T polymorphism may affect ischemic stroke through plasma lipid and lipoprotein levels.

Brain Ischemia ; blood ; genetics ; metabolism ; Cholesterol, LDL ; genetics ; metabolism ; Female ; Genetic Predisposition to Disease ; Humans ; Integrin alpha2 ; genetics ; metabolism ; Integrin beta3 ; genetics ; metabolism ; Lipid Metabolism ; genetics ; Lipids ; blood ; Male ; Middle Aged ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide

To explore the correlation between the C807T polymorphism of platelet membrane glycoprotein I a (GP I a) gene and aspirin resistance in Chinese people, 200 patients with high-risk of atherosclerosis took aspirin (100 mg/d) for 7 days. Platelet aggregation function was detected using adenosine diphosphate (ADP) and arachidonic acid (AA) before and after the administration of aspirin. Then the subjects were divided into three groups according to the results of platelet aggregation function: an aspirin resistant (AR) group, an aspirin semi-responder (ASR) group and an aspirin-sensitive (AS) group. Platelet GP I a gene 807CT polymorphism was examined by means of polymerase chain reaction-sequence specific primers (PCR-SSP). The results showed that T allelic frequency in AR group and ASR group were higher that of AS group (P<0.005), and the prevalence of genotypes (TT+TC) of these two groups was significantly higher than that in AS group (P<0.05). Platelet GP I a T allele was significantly associated with aspirin resistance as revealed by multiple logistic regression (OR=3.76, 95% CI: 2.87-9.58). The results suggest that inherited platelet GP I a variations may have an important impact on aspirin resistance and the presence of GP I a T allele may be a marker of genetic susceptibility to aspirin resistance.
Aspirin/*administration & dosage ; Atherosclerosis/drug therapy ; Atherosclerosis/genetics ; Drug Resistance/*genetics ; Integrin alpha2/*genetics ; Platelet Aggregation Inhibitors/*administration & dosage ; Polymorphism, Genetic/*genetics