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

The purpose of this paper is to investigate the probable molecular mechanism in mechano-transduction of the regulation of integrins and the effects of cyclic biaxial mechanical strain on proliferation and synthetic function in the osteoblasts isolated from 3-month-old female Sprague-Dawley (SD) rats. The osteoblasts were cultured in F-12 medium contained with 10% fetal bovine serum(FBS) and grown to subconfluency in Flexercell type I dishes in a humidified incubator with 5% CO2 and 95% air at 37 degrees C. Mechanical strains were applied to the cells for periods of 30 min, 2, 4 and 8 hours every day, lasting 2 days. The amplitude of mechanical strain applied to the cells were 400, 1,000 and 4,000 mu strain respectively, at a frequency of one hertz(1 Hz). Unstrained cells were used as control. The expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts and proliferation activity of osteoblasts were studied with Flow Cytometry(FCM). The content of osteocalcin, carboxyterminal propeptide of type-I procollage(PICP), total protein secreted by osteoblastes were detected with the isotope labelling method. The results showed that there are actual expressions of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts without mechanical strain and that the expression of integrins beta 1 is highest. The mechanical strain increased the expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts, but the strain-related up-regulation of expression of integrins alpha 2, beta 1, beta 3 are different in various amplitude and different duration of mechanical stains. The up-regulation of expression of integrins beta 3 is most sensitive to mechanical strain. The up-regulation of expression of integrins alpha 2, beta 1, beta 3 is higher at 4,000 mu strain than at 400, 1,000 mu strain. The mechanical strain can elevate the proliferation activity and the synthetic function of osteoblast at 400, 1,000 mu strain. However, the mechanical strain increased significant the proliferation in the osteoblasts and suppressed obviously the synthetic function in the osteoblasts. In the present study, the reaction of the osteoblasts in 3 month-old rat to the mechanical stimulation suggested that 1) expressions of integrins alpha 2, beta 1, beta 3 were increased in a amplitude of strain-dependent manner; 2) the changes of expression of integrins alpha 2, beta 1, beta 3 relate close to the changes of the proliferation and synthetic function of the osteoblasts. Low amplitude of strain can increase the proliferation and the synthetic function of the osteoblasts along with up-regulation of expression of integrins alpha 2, beta 1, beta 3; while higher amplitude of strain elevated significantly the proliferation of osteoblasts and suppressed obviously the synthetic function of the osteoblasts along with up-regulation of expression of integrins alpha 2, beta 1, beta 3. The amplitude of 4,000 mu strain is an optimal amplitude as stimulus for up-regulation of expression of integrins alpha 2, beta 1, beta 3 on the membrane of osteoblasts and increase the proliferation activity, but decrease the synthetic function of osteoblasts in the present study. Accordingly it indicates that integrins have a important role in regulation of signal transduction pathway in osteoblasts as a result of mechanical strain.
Animals ; Cell Division ; Cells, Cultured ; Female ; Integrin alpha2 ; biosynthesis ; Integrin beta1 ; biosynthesis ; Integrin beta3 ; biosynthesis ; Mechanics ; Osteoblasts ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley

BACKGROUND: We have previously shown that the vimentin-positive subset of human breast cancer cell lines, which are more invasive in vitro and in vivo, can activate MMP-2 when cultured on vitrogen gels whereas the poorly invasive, poorly metastatic vimentin-negative subset cannot (J. Cell Physiol. 150: 534, 1992). METHODS: In this study, we compared the interaction of human breast cancer cell lines from each group with respect to the interaction with vitrogen. RESULTS: The cell lines capable of responding to vitrogen for MMP-2 activations showed an enhanced ability to attach to vitrogen-coated culture wells. MCF-7 (non-MMP-2 activating) and MDA-MB-231 (MMP-2 activating) cells were selected for more detailed analysis. MDA-MB-231 cells showed a greater affinity for the 3-dimensional vitrogen than MCF-7 cells. Attachment of both lines to thin coatings of vitrogen was shown to require divalent cations and to be mediated by beta1 integrins. The alpha5 subunit, however, was shown to be involved in fibronectin attachment, but not vitrogen attachment. The GRGDSP peptide dramatically inhibited fibronectin attachment of both cell lines, but did not effect the vitrogen attachment of either. In contrast, the KGDEA recognition sequence for alpha2 integrin inhibited the attachment of MDA-MB-231 cells to vitrogen, but not to fibronectin or laminin. CONCLUSIONS: These results show that the subset of human breast cancer cell lines which respond to the vitrogen gel with MMP-2 activation may interact more easily with the vitrogen, apparently through integrin-mediated recognition. Preliminary analysis reveals that the alpha2beta1 integrin, previously implicated in vitrogen interaction may mediate this response.
Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Cations, Divalent ; Cell Line* ; Fibronectins ; Gels ; Humans* ; Integrin alpha2 ; Laminin ; MCF-7 Cells

OBJECTIVEPlatelet membrane glycoprotein (GP) Ia/IIa complex is the major collagen receptor on platelets. Platelet activation by GP Ia/ IIa dependent adhesion leads to cellular events that catalyze prothrombin conversion and fibrin clot formation. Correlation between the polymorphism of platelet membrane GP Ia gene and myocardial infarction (MI) was explored.

METHODSA total of 137 patient s with myocardial infarction and 175 controls with no history of coronary heart disease, thrombogenic and hemorrhagenic diseases were studied by case-control. Platelet GP I a gene 807 C/T polymorphisms were checked by polymerase chain reaction-sequence specific primers.

RESULTSThere were significant differences in the distribution of T and C alleles between MI and control groups (T:42.70% vs 32.00%, C:57.30% vs 68.00%, P<0.001). No matter among all subjects or among subjects aged

CONCLUSIONThe above data suggest that there is a strong association between the presence of GP Ia T allele and MI. T allele ca n be a marker of genetic susceptibility to MI. These need to be substantiated by a large scale and prospective study.

Aged ; Alleles ; Female ; Gene Frequency ; Genotype ; Humans ; Integrin alpha2 ; genetics ; Male ; Middle Aged ; Myocardial Infarction ; genetics ; pathology ; Polymorphism, Genetic

PURPOSE: To investigate the effect of ionizing radiation on high mucin-producing colon cancer cells, we evaluated homotypic cell adhesion, cell-matrix adhesion, and matrix metalloproteinases (MMPs) on HM7 cells. METHODS: After an irradiation of 60 Gy for 48 hours on HM7 cells, we evaluated cellular proliferation, colony-forming ability, homotypic adhesion, cell-matrix binding, and integrin subunit expressions. Also, alterations of MMPs expression were analyzed by using zymography. RESULTS: Cell proliferation of HM7 colon cancer cells was not remarkably affected even after high doses of radiation; however, clonogenic cell growth was significantly affected. Homotypic cell-cell adhesion and cell adhesion to ECM components and basement membrane protein matrigel were significantly increased after irradiation. Radiation induced expressions of cell surface integrin alpha2, alpha3, and beta1 subunits of HM7 cells. The activities of secreted MMPs (MMP-9 and MMP-2) were remarkably inhibited by radiation. CONCLUSIONS: These finding suggest the biologic characteristics of high-mucin-producing colorectal carcinomas. Even though the radiation-associated cellular alterations of HM7 cells with or without matrix proteins were not remarkably different from other cancer cell types studied, the radio-resistant behavior of high mucin producing HM7 cells may explain the aggressive characteristics of mucinous colorectal carcinomas.
Basement Membrane ; Cell Adhesion* ; Cell Proliferation ; Colon* ; Colonic Neoplasms* ; Colorectal Neoplasms ; Integrin alpha2 ; Matrix Metalloproteinases* ; Mucins* ; Population Characteristics ; Radiation, Ionizing*

PURPOSE: Integrins are cell surface proteins that anchor the cells to the extra-cellular matrix. It has recently been found that integrins are involved in proliferations, migration, differentiation and survival signal transduction. We studied the expression of integrins in normal and cancer tissue of Korean breast cancer patients, and investigated the relationship between integrin expression and the characteristics of breast cancer. METHODS: Normal and malignant breast tissues were taken from 25 breast cancer patients who were admitted to the Ajou University Hospital. Specimens were immediately preserved in a nitrogen tank at the time of the operation. Total RNA was extracted, and semi-quantitative reverse transcriptase polymerase chain reactions (RT-PCR) performed with PCR primers for integrin alpha1, alpha2, alpha5, and alphav, and integrin beta1, and beta3. The integrin expressions were compared between the normal and malignant tissues, and the expressions were analyzed in relation to tumor characteristics. RESULTS: Integrin alpha1, alpha5, alphav, beta1, and beta3 were significantly over-expressed in breast cancer tissue than in normal tissue. There was no difference in integrin alpha2 expression between the normal and cancer tissues. Integrin beta1 was over-expressed to a greater extent in lower histological grade carcinomas and to a lesser extent in high grade tumors. Hormonal receptor positive tumor tissue had more alphav, alpha5, and beta1 integrin expressions. There was no significant relationship between integrins and tumor size, lymph node meta-stasis, lymphovascular involvement, or c-erb-B2 expression. CONCLUSIONS: Integrins alpha1, alpha5, alphav and beta3 were over- expressed in malignant breast tissue to a greater extent than in normal tissue. However, studies on the localization of integrin expression in cancer tissue, and co-relations of integrin over-expressions, with survival and drug sensitivity, must be followed to evaluate the clinical value of integrin expression.
Antigens, CD29 ; Breast Neoplasms* ; Breast* ; Humans ; Integrin alpha1 ; Integrin alpha2 ; Integrins* ; Lymph Nodes ; Membrane Proteins ; Nitrogen ; Polymerase Chain Reaction ; RNA ; RNA-Directed DNA Polymerase ; Signal Transduction

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

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

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

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