Effects of the ITGA2B Nonsense Mutation (c.2659C > T, p.Q887X) on Platelet Function in a Mouse Model of Glanzmann's Thrombasthenia Generated with CRISPR/Cas9 Technology / 中国实验血液学杂志

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.

Memantine Attenuates Salicylate-induced Tinnitus Possibly by Reducing NR2B Expression in Auditory Cortex of Rat

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.

Proposal of conditional random inter-stimulus interval method for unconstrained enclosure based GPIAS measurement systems

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.

Effect of the Integrin β3 Cytoplasmic NITY Motif on α II bβ3-Mediated Cell Functions in CHO Cell Model / 中国实验血液学杂志

<p><b>UNLABELLED</b>OBJLECTIVE: To investigate the effect of integrin β3 cytoplasmic NITY motif on αIIbβ3-mediated cell functions.</p><p><b>METHODS</b>Stable 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.</p><p><b>RESULTS</b>CHO-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.</p><p><b>CONCLUSION</b>Deletion 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.</p>

Induction of Selective Cell Death of Oral Squamous Carcinoma Cells by Integrin alpha2 Antibody and EGFR Antibody

Role of IL-1alpha in Cisplatin-Induced Acute Renal Failure in Mice

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.

Identification of compound heterozygous mutations in the ITGA2B gene in a Chinese patient with Glanzmann thrombasthenia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Glanzmann 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>The 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.</p>

Association of the integrin gene polymorphisms with ischemic stroke and plasma lipid levels / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Polymerase 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.</p><p><b>RESULTS</b>Plasma 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).</p><p><b>CONCLUSION</b>The 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.</p>

Effects of WWOX on ovarian cancer cell attachment in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action.</p><p><b>METHODS</b>Attachment 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.</p><p><b>RESULTS</b>Attachment 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.</p><p><b>CONCLUSION</b>WWOX 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.</p>

Association of the platelet membrane glycoprotein I a C807T gene polymorphism with aspirin resistance / 华中科技大学学报（医学）（英德文版）

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.

Effect of Ionizing Radiation on Homotypic Cell Adhesion, Cell-Matrix Adhesion, Matrix Metalloproteinases Excretion of High Mucin Producing HM7 Colon Cancer Cells

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.

Genotype and allele frequencies of the platelet glycoprotein genes associated with arterial thrombosis in Korean population / 대한수혈학회지

BACKGROUND: Ischemic heart disease and cerebrovascular disease are the main causes of death and platelets are responsible for the formation of arterial thrombi. Platelet membrane glycoproteins (GP) associated with coagulation pathway are GPIb/V/IX, GPIa/IIa, and GPIIb/IIIa. We evaluated genotype and allele frequencies of seven platelet glycoprotein genes associated with arterial thrombosis. METHODS: Genomic DNA was isolated from peripheral blood of 300 unrelated Korean and single nucleotide polymorphism of platelet glycoproteins was analyzed. PCR with sequence specific primers was used to investigate GPIa C807T and GPIbalpha VNTR polymorphism. PCR-RFLP (restriction fragment length polymorphism) was used to investigate GPIa G1648A and C2531T, GPIbalpha C524T and T-5C, and GPIIIa T1565C polymorphism. RESULTS: The allele frequencies of GPIa C807T were 807C 0.733, 807T 0.267; GPIa 1648G 0.975, 1648A 0.025; GPIa C2531T, 2531C 1.000, 2531T 0.000; GPIbalpha C524T, 524C 0.927, 524T 0.073; GPIbalpha VNTR, A 0.017, B 0.015, C 0.558, D 0.410; GPIbalpha T-5C, -5T 0.726, -5C 0.274; GPIIIa T1565C, 1565T 0.995, 1565C 0.005. CONCLUSION: The genotype and allele frequencies of GPIa G1648A, GPIbalpha C524T, and GPIIIa T1565C were similar to established data. GPIa 807T and -5T allele of Kozak polymorphism showed low frequency compared with other ethnic group. Allele frequencies of GPIbalpha VNTR A and B alleles were very alike (0.017 vs 0.015). In this study, we firstly evaluated the genotype and allele frequencies of GPIa C2531T and GPIbalpha VNTR, T-5C polymorphisms in Korean population. This study will serve as a basic data for the study of platelet glycoproteins associated with arterial thrombosis in Korean.

Effects of Ovariectomy on Bone Mineral Density and Integrin Expression in Maxilla of Rats / 대한해부학회지

In postmenopausal osteoporosis, estrogen deficiency leads to unbalance of bone metabolism, decreased bone formation and increased bone resorption, and the result is reduced bone mineral density (BMD) and bone stiffness. The processes of bone formation and resorption involves the expression of integrins in anchoragedependent cells, such as osteoblast and osteoclast. The osteoporosis-induced rats frequently demonstrated the loss of trabecular bone volume in the tibia, vertebra and mandible due to estrogen depletion. However, in maxilla, study has been rare because of its anatomical limits. So the objective of this study was to investigate bony change and property of integrin expression in maxilla of osteoporosis-induced rats. 12-week-old female Sprague-Dawley rats were bilaterally ovariectomized (OVX). At 1, 2, 3, 4, 8 and 12 weeks, control and OVX group rats were sacrificed respectively. BMD of maxilla of the rats was measured using dual-energy X-ray absorptiometry (DEXA). And then the histopathologic observation, histomorphometric analysis and immunohistochemistry with CD44, alpha2 integrin, alpha5 integrin, alpha6 integrin, alphav integrin and beta3 integrin were done. BMD of alveolar bone in maxilla was decreased with significance statistically after OVX 4 weeks and was decreased 18.15% at OVX 12 weeks group compared to control group. From OVX 4 to 12 weeks, the thickness of periodontal ligament space was decreased, the number of osteoclast and the size of marrow stroma were increased than control group. By histomorphometric analysis, the size of marrow stroma of alveolar bone in maxilla was increased 86.42% at OVX 12 weeks group compared to control group. CD44 was widely expressed throughout the odontoblast, cementoblast, dental pulp, preiodontal ligament, osteocyte, osteoclast and perivascular tissue at control group, and CD44 immunoreactivity was increased the odontoblast, osteoblast and osteoclast at OVX groups. alpha2 integrin was expressed the odontoblast and osteoblast at control group, but alpha2 integrin immunoreactivity was decreased the osteoblast at OVX 12 weeks group. alpha5 integrin was expressed the cementoblast, osteoblast and osteoclast at control group, and alpha5 integrin immunoreactivity was decreased the osteoblast and was increased the osteoclast from OVX 4 weeks group. alpha6 integrin was weakly expressed the odontoblast, cementoblast, osteoblast and osteoclast at control group, and alpha6 integrin immunoreactivity was weakly increased the osteoclast from OVX 4 weeks. alphav integrin was expressed the odontoblast and osteoclast at control group, and alphav integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. beta3 integrin was expressed the osteocyte and osteoclast at control group, and beta3 integrin immunoreactivity was strongly increased the osteoclast from OVX 4 weeks. From these results, alveolar bone in maxilla of OVX rats was decreased BMD gradually. Moreover, alpha2 and alpha5 integrin expression of osteoblast was decreased, and alpha5, alphav and beta3 integrin expression of osteoclast was increased in OVX rats. Thus, this study indicates that consideration of reduced BMD is necessary in dental procedure of postmenopausal women.

Correlation between the polymorphism of glycoprotein Ia gene and acute coronary syndrome / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>The platelet membrane glycoprotein (GP) Ia/IIa plays a major part as a primary collagen receptor in platelet function. Previous studies indicated that variations of GPIa/IIa density and function are associated with the 807 C/T polymorphism of GPIa gene in American and Spanish Caucasian populations. This study investigated the correlation between acute coronary syndrome (ACS) and 807 C/T dimorphism of GPIa gene in Chinese of Han ethnicity.</p><p><b>METHODS</b>A case-control study was carried out, including 75 patients with either acute myocardial infarction (AMI) or unstable angina pectoris (UAP), and 65 controls with no history of coronary heart disease, thrombogenic and hemorrhagenic diseases. Genotypes of GPIa were checked by polymerase chain reaction-sequence specific primers (PCR-SSP) technique.</p><p><b>RESULTS</b>The frequencies of both homozygotes and heterozygotes for T807 allele (TT+TC) were significantly higher in patients with AMI than in controls (62.16% vs 33.85%, P<0.01; odds ratio 3.21). The prevalence of (TT+TC) genotypes was also markedly higher in patients with UAP than in controls (65.79% vs 33.85%, P < 0.005; odds ratio 3.76). There was significant difference in the distribution of (TT+TC) genotypes not only between all patients and controls (64.00% vs 33.85%, P<0.005; odds ratio 3.47) but also between the two subgroups aged <60 years (70.00% vs 38.24%, P<0.005; odds ratio 3.77). However, there was no significant difference in the distribution of (TT+TC) genotypes between patients with AMI and with UAP. Platelet GPIa T807 allele remained significantly associated with AMI and UAP by multiple logistic regression (odds ratio 4.94).</p><p><b>CONCLUSION</b>This study suggests a strong association between presence of GPIa T807 allele and ACS. T807 allele can be a marker of genetic susceptibility to ACS.</p>

Association of the Gene Polymorphisms of Platelet Glycoprotein Ia and IIb/IIIa with Myocardial Infarction and Extent of Coronary Artery Disease in the Korean Population

Platelet membrane receptor glycoproteins (GP) are essential for the platelet activation process, and the genetic polymorphisms in the genes that encode platelet glycoproteins have been proposed to influence the risk of acute coronary syndrome and atherosclerosis. In this study, we investigated the role of GPIa, HPA-1 and HPA-3 polymorphisms as putative risk factors for myocardial infarction (MI) and the extent of coronary artery disease. We selected 1, 073 subjects who underwent coronary angiography; 242 had normal or minimal coronary atherosclerosis, and 831 patients had significant coronary artery disease (CAD). The genotype was determined by the methods of single base extension for C807T/G873A polymorphisms of GPIa, and restriction fragment length polymorphism for HPA-1 and HPA-3. The C807T and G873A polymorphisms of GPIa showed complete linkage in the Korean population. For HPA-1 gene polymorphism, only the HPA-1a/a (PlA1/A1) genotype was observed in 192 selected subjects from our study population. The distribution of GPIa (C807T/G873A) and HPA-3 genotypes did not differ significantly between normal subjects and CAD subjects. No significant association between MI and both gene polymorphisms was present. However, for the subgroup analysis of young male patients whose age was less than 56 years, the genotype frequency of HPA-3b/b was significantly lower in patients with MI compared to patients without a history of MI (7.5% vs. 20.0%, p=0.04). The odds ratio for HPA-3 b homozygosity versus the HPA-3a carrier was 0.32 (95% CI, 0.10- 0.99, p=0.04). Conclusively, HPA-3 polymorphism was associated with MI in Korean individuals younger than 56 years of age, but other polymorphisms of GP, which we studied, were not associated with both the extent of coronary atherosclerosis or MI.

The effect of mechanical stimulation on the expression of alpha 2, beta 1, beta 3 integrins and the proliferation, synthetic function in rat osteoblasts / 生物医学工程学杂志

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.

Association of the polymorphism of platelet membrane glycoprotein I a gene with myocardial infarction / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Platelet 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>There 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 <or= 60 years, the prevalence of genotypes (TT+TC) in MI group was significantly higher than that in control group [in all subjects; 69.34% vs 51.43%, P<0.005, odds ratio(OR)=2.14, 95% CI: 1.34-3.41; in subjects aged <or= 60 years; 75.90% vs 51. 52%, P<0.005, OR=2.96,95% CI 1.58-5.55]. Platelet GP Ia T allele was significantly associated with MI by multiple logistic regression (OR=4.96, 95% CI:2.55-10.90).</p><p><b>CONCLUSION</b>The 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.</p>

The Expression of Integrins in Korean Breast Cancer Patients

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.

The Studies for Interaction of MMP-2 Activating and Nonactivating Human Breast Cancer Cell Lines with Vitrogen

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.