Tsuyoshi Nakazawa

A potential role for α-actinin in inside-out αIIbβ3 signaling.

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.

Bleeding tendency and impaired platelet function in a patient carrying a heterozygous mutation in the thromboxane A2 receptor.

Summary.  Background: Thromboxane A2 receptor (TXA2R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA2R abnormality and investigate the mechanism for the impaired response to TXA2. Patient: The proband (OSP‐2, PT) was a 7‐year‐old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm)‐induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA2R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA2R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA2R. We then examined αIIbβ3 activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained αIIbβ3 and Rap1B activation in the proband. In addition, platelet secretion as monitored by P‐selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y12 has been shown to play a critical role in the sustained αIIbβ3 activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 μm) partially restored the sustained αIIbβ3 activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA2R mutation is, at least in part, as a result of the decrease in ADP secretion.

Antifungal Activity of Micafungin in Serum

ABSTRACT We have evaluated the antifungal activity of micafungin in serum by using the disk diffusion method with serum-free and serum-added micafungin standard curves. Serum samples from micafungin-treated patients have been shown to exhibit adequate antifungal activity, which was in proportion to both the applied dose and the actual concentration of micafungin measured by high-performance liquid chromatography. The antifungal activity of micafungin in serum was also confirmed with the broth microdilution method.

Donor Cell-Derived Chronic Myeloproliferative Disease with t(7;11)(p15;p15) after Cord Blood Transplantation in a Patient with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

We report a case of donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) occurring after cord blood transplantation (CBT). A 41-year-old man developed precursor B-cell acute lymphoblastic leukemia with a karyotype of 46, XY, t(9;22)(q34;q11) and inv(9)(p11;q13), for which he received CBT from a sex-mismatched donor at the first complete remission of the leukemia. Five months after CBT, gradual neutrophilia of unknown origin developed following the myeloid reconstitution after CBT. Karyotyping of bone marrow cells at 9 months after CBT showed 46,XX, t(7;11)(p15;p15) in 17/20 dividing cells, but neither Philadelphia chromosome (Ph) nor inv(9)(p11;q13) was present. This is the first report of chronic myeloproliferative disease with t(7;11)(p15;p15) that developed in donor cells after CBT. The donor was well-developed and healthy, at least at the time of follow-up, half a year after the birth.

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14–22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40‐transfected mortal, immortalized, anchorage‐independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild‐type even in the malignant cell lines, because the expression and the LT‐binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14–22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage‐independent cell line suppressed its anchorage‐independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14–22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47–53, 1999.

A Novel Chromosomal Abnormality, t(6;10)(q27;q22), Found in a Polycythemic Potential Donor for Allogeneic Hematopoietic Stem Cell Transplantation

A 59-year-old man was a potential donor for allogeneic hematopoietic stem cell transplantation and was found to be healthy but slightly polycythemic. The bone marrow was morphologically normal, but karyotyping of bone marrow cells showed t(6;10)(q27;q22) in 7 of 20 metaphases analyzed by G-banding and only the t(6;10) abnormality in 3 of 5 metaphases analyzed by the spectral karyotyping method. G-banding analysis of peripheral blood mononuclear cells cultured with phyto-hemagglutinin for 72 hours showed a normal karyotype in all 20 metaphases analyzed. These findings suggest clonal expansion with t(6;10)(q27;q22) in the bone marrow of this individual. He was determined to be ineligible for donation. A coordinated nationwide work-up for older donors is necessary to ensure high-quality standards.