Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages.

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

Virtual Active Touch: Perception of Virtual Gratings Wavelength through Pointing-Stick Interface.

Tactile feedback enhances the usability and enjoyment of human-computer interfaces. Many feedback techniques have been devised to present tactile stimuli corresponding to a user's hand movements taking account of the concept of active touch. However, hand movements may not necessarily be required for achieving natural tactile feedback. Here, we propose a virtual-active-touch method that achieves haptic perception without actual/direct hand movements. In this method, a cursor manipulated by a force-input device is regarded as a virtual finger of the operator on the screen. Tactile feedback is provided to the operator in accordance with cursor movements. To validate the translation of virtual roughness gratings, we compare the virtual-active-touch interface with an interface that involves actual hand movements. By using the appropriate force-to-velocity gain for the pointing-stick interface, we show that the virtual-active-touch method presents the surface wavelengths of the gratings, which is a fundamental property for texture roughness, and that the gain significantly influences the textures experienced by the operators. Furthermore, we find that the perceived wavelengths of objects scaled and viewed on a small screen are skewed. We conclude that although some unique problems remain to be solved, we may be able to perceive the surface wavelengths solely with the intentions of active touch through virtual-active-touch interfaces.

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

BACKGROUND: Thromboxane A(2) receptor (TXA(2)R) abnormality appears to dominantly disturb platelet function. OBJECTIVES: To reveal a molecular genetic defect in a patient with TXA(2)R abnormality and investigate the mechanism for the impaired response to TXA(2). 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 TXA(2)R 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 TXA(2)R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA(2)R. We then examined α(IIb)ß(3) activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α(IIb)ß(3) 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 P2Y(12) has been shown to play a critical role in the sustained α(IIb)ß(3) activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 µm) partially restored the sustained α(IIb)ß(3) 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 TXA(2)R mutation is, at least in part, as a result of the decrease in ADP secretion.

Vibrotactile Stimuli Applied to Finger Pads as Biases for Perceived Inertial and Viscous Loads.

The perception of the mass and viscosity of an object is based on the dynamic forces applied to our hands when we jiggle or lift the object [1], [2], [3]. This force is commonly assumed to be sensed by kinetic receptors [4] in our muscles or tendons. When jiggling objects, we also experience the cutaneous deformation of our finger pads. In this study, we show that the dynamic vibration on the finger pad influences our perception of mass and viscosity. We experimentally confirm that the vibration on the finger pad, that synchronizes with the hand's accelerations or velocities, enhances the perceived changes in the mass or viscosity when the vibrotactile stimuli and the changes in the mass and viscosity are in the same perceptual direction. For example, when the increased mass and an acceleration-synchronized tactile stimulus-which is a positive bias for the mass-are simultaneously presented to the experiment participants, they respond that the perceived increase in the mass is enhanced. In contrast, when the tactile and proprioceptive stimuli are in perceptually opposite directions, the vibrotactile stimuli cancel the perceived changes in the mass and viscosity. In particular, the effect of the velocity-synchronized vibration on perception is stronger than the effect of the actual viscosity.

Detectability and Perceptual Consequences of Delayed Feedback in a Vibrotactile Texture Display.

This study estimated the maximum allowable system latency for haptic displays that produce tactile stimuli in response to the hand movements of users. In Experiment 1, two types of detection thresholds were estimated for the time delay of stimuli through psychophysical experiments involving 13 participants. One was a threshold for the users to notice the existence of a time delay. The other was a threshold for the users to experience changes in the perceived textures in comparison with stimuli with no time delay. The estimated thresholds were approximately 60 and 40 ms, respectively. In interviews, the participants reported that they experienced various types of subjective changes due to the time delay. In Experiment 2, the types of subjective sensations that might be altered by the time delay were investigated. The time delays were controlled based on the acceleration of the hand motions of the participants. The participants evaluated the differences in the perceived textures between the stimuli with a controlled time delay and ones with no delay. The results indicated that the participants associated the time-delayed stimuli with changes in mechanical parameters such as kinetic friction coefficient in addition to changes in the perceived roughness of the textures.

Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis.

OBJECTIVE AND DESIGN: We monitored the membrane fusion of liposomes to determine if the minimal components of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE), which is involved in mast cell exocytosis, have fusogenic activity. METHODS: Three core components of SNARE were reconstituted into liposomes. Membrane fusion between liposomes containing vesicle associated membrane protein (VAMP) -7 or -8 and liposomes containing synaptosomal-associated protein 23 kDa (SNAP23) and syntaxin-3 or -4 was monitored by fluorescence resonance energy transfer. RESULTS: The combination of SNAP23/syntaxin-3/VAMP-8 showed the most efficient liposome-liposome fusogenic activity. Liposomes with VAMP-7 exhibited poor fusogenic activity regardless of the syntaxin isoform. CONCLUSION: The core components of SNAP23, syntaxin-3, and VAMP-8 appear to be minimal machinery to induce membrane fusion, while VAMP-7 appears to be unessential for membrane fusion.

Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.

Analyses of genetic abnormalities in type I CD36 deficiency in Japan: identification and cell biological characterization of two novel mutations that cause CD36 deficiency in man.

To elucidate genetic abnormalities in type I CD36 deficiency, we analyzed 28 Japanese subjects whose platelets and monocytes/macrophages lacked CD36 on their surface. We identified two novel mutations in the CD36 gene. One was a complex deletion/insertion mutation, in which 3 bp, GAG, were deleted at nucleotide (nt) 839-841, and 5 bp, AAAAC, were inserted at the same position (839-841del-->insAAAAC). Mutation 839-841del-->insAAAAC led to a frameshift and appearance of a premature stop codon; it was also accompanied with a marked reduction in the amount of CD36 mRNA. The other was a 12-bp deletion at nt 1438-1449 (1438-1449del) accompanied with or without skipping of exon 9 (nt 959-1028). Mutation 1438-1449del led to an inframe 4-amino-acid deletion, whereas exon 9 skipping led to a frameshift and the appearance of a premature stop codon. Expression assay revealed that both 1438-1449del and exon 9 skipping directly caused impairment of the surface expression of CD36. A survey of the five known mutations including 839-841del-->insAAAAC and 1438-1449del in type I CD36-deficient subjects demonstrated that the five mutations covered more than 90% of genetic defects among them and that the substitution of T for C at nt 478 (478C-->T) was the most common mutation with more than 50% frequency. However, none of the four subjects that possessed isoantibodies against CD36 had 478C-->T, suggesting that 478C-->T prevents the production of isoantibodies against CD36.

Anti-alphavbeta3 antibodies in chronic immune thrombocytopenic purpura.

In chronic immune thrombocytopenic purpura (ITP), anti-GPIIb-IIIa (alphaIIbbeta3) autoantibodies have been detected in serum and/or platelet-associated IgG (PAIgG) and considered as one of the major causes. We examined whether anti-alphavbeta3 antibodies might be present in ITP cases because of the similarity between alphavbeta3 and GPIIb-IIIa (alphaIIbbeta3). Modified antigen capture ELISA (MACE) using human umbilical vein endothelial cells (HUVEC) showed the presence of serum anti-alphavbeta3 antibodies in 23 of 80 ITP patients (29%). Cross-adsorption studies between platelets and HUVEC demonstrated that most of anti-alphavbeta3 and anti-GPIIb-IIIa antibodies exclusively reacted with alphavbeta3 and GPIIb-IIIa, respectively. Platelet-associated anti-GPIIb-IIIa antibodies did not react with alphavbeta3, either. Interestingly, patients having anti-alphavbeta3 antibodies showed significantly lower platelet counts than negative patients. These results indicate the serum anti-alphavbeta3 antibodies are different ones from the classical anti-GPIIb-IIIa (alphaIIbbeta3) antibodies and would provide a new insight into the pathophysiology of ITP as well as the autoantigenic epitopes on beta3 integrins.

Ligand binding to integrin alpha(v)beta(3) requires tyrosine 178 in the alpha(v) subunit.

Integrin alpha(v)beta(3) has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in alpha(v), a non-I-domain alpha subunit, remain to be identified. Recently in alpha(IIb), the other partner of the beta(3) subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of alpha(v) in ligand binding. Wild-type alpha(v)beta(3) was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl(2)) or a mutation of beta(3) threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of alpha(v) abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aalpha(v) also failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-alpha(v)beta(3) antibody, and the expression of beta(3) ligand-induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with alpha(IIb), none of the mutations in the predicted W3 4-1 loop in alpha(v) impaired ligand binding. These results implicate alpha(v) Y178 in ligand binding to alpha(v)beta(3), and they suggest that there are key structural differences in the adhesive ligand-binding sites of alpha(v)beta(3) and alpha(IIb)beta(3).

[Comparison of reticulated platelet count with plasma glycocalicin concentration as a marker of platelet turnover in patients with thrombocytopenic disorders].

Measurements of plasma glycocalicin (GC) and reticulated platelets (RP) have been reported to be useful for classifying thrombocytopenic disorders. However, there have been no reports comparing the clinical usefulness of the two methods. We measured GC and RP levels simultaneously in 39 patients with idiopathic thrombocytopenic purpura (ITP), 15 patients with aplastic anemia (AA), and 17 patients with hypoplastic thrombocytopenia (HypoT) due to chemotherapy. The GC index (GC level normalized for the individual platelet count) and the percentage of RP (%RP), a parameter of platelet life span, were very high (7.5 +/- 11.4 and 20.8 +/- 13.0%, respectively) in patients with ITP as compared with those of healthy subjects (1.3 +/- 0.5 and 7.9 +/- 2.5%, respectively). However, 6 AA patients and 14 HypoT patients, in whom platelet life span is thought to be normal, also had an elevated GC index, suggestive of a false positive result. The RP, a parameter of platelet production, was low in all AA and HypoT patients except for one in each case. However, the GC level, an additional parameter of platelet production, was normal in 4 AA and 8 HypoT patients, indicating that it is not a sensitive indicator. We conclude that the RP and %RP are more feasible markers of thrombopoiesis and platelet life span, respectively, than the GC level and GC index.

The 2-phenylbenzotriazole-type water pollutant PBTA-2 has cytochalasin B-mimetic activity.

The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix). In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ. In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells. Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced. We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells. A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells. Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B. Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate. The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far. This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations.

Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering.

Several scaffold proteins for neurotransmitter receptors have been identified as candidates for receptor targeting. However, the molecular mechanism underlying such receptor clustering and targeting to postsynaptic specializations remains unknown. PSD-Zip45 (also named Homer 1c/vesl-1L) consists of the NH(2) terminus containing the enabled/VASP homology 1 domain and the COOH terminus containing the leucine zipper. Here, we demonstrate immunohistochemically that metabotropic glutamate receptor 1alpha (mGluR1alpha) and PSD-Zip45/Homer 1c are colocalized to synapses in the cerebellar molecular layer but not in the hippocampus. In cultured hippocampal neurons, PSD-Zip45/Homer1c and N-methyl-D-aspartate receptors are preferentially colocalized to dendritic spines. Cotransfection of mGluR1alpha or mGluR5 and PSD-Zip45/Homer 1c into COS-7 cells results in mGluR clustering induced by PSD-Zip45/Homer 1c. An in vitro multimerization assay shows that the extreme COOH-terminal leucine zipper is involved in self-multimerization of PSD-Zip45/Homer 1c. A clustering assay of mGluRs in COS-7 cells also reveals a critical role of this leucine-zipper motif of PSD-Zip45/Homer 1c in mGluR clustering. These results suggest that the leucine zipper of subsynaptic scaffold protein is a candidate motif involved in neurotransmitter receptor clustering at the central synapse.

CD36 mediates long-chain fatty acid transport in human myocardium: complete myocardial accumulation defect of radiolabeled long-chain fatty acid analog in subjects with CD36 deficiency.

Long-chain fatty acids (LCFA) are the major energy substrate for heart and their oxidation is important for achieving maximal cardiac work. However, the mechanism of uptake of LCFA by myocardium has not been clarified. We previously reported that bovine myocardial LCFA transporter has a sequence homology to human CD36. Clinically, total defect of myocardial uptake of radiolabeled long-chain fatty acid analog [123I-BMIPP: Iodine-123 15-(p-iodophenyl)-(R,S)-methylpentadecanoic acid] has been reported in some restricted cases, but the etiology has not been clarified. In the present study, we analyzed CD36 expression and CD36 gene in subjects who showed total lack of myocardial 123I-BMIPP accumulation, and, vice versa, evaluated myocardial 123I-BMIPP uptake in subjects with CD36 deficiency. Four unrelated subjects were evaluated, Two were found to have negative myocardial LCFA accumulation by 123I-BMIPP scintigraphy, after which the expression of CD36 on their platelets and monocytes was analyzed. Remaining two subjects were identified as CD36 deficiency by screening, then 123I-BMIPP scintigraphy was performed. Expression of CD36 on platelets and monocytes was measured by flow cytometric analysis. The molecular defects responsible for CD36 deficiency was detected by allele-specific restriction enzyme analysis. CD36 expression was totally deficient in all 4 subjects on both platelets and monocytes. Two subjects were homozygous for a 478C-->T mutation. One was heterozygous for the dinucleotide deletion of exon V and single nucleotide insertion of exon X, and remaining one was considered to be heterozygous for the dinucleotide deletion of exon V and an unknown gene abnormality. All cases demonstrated a completely negative accumulation of myocardial LCFA despite of normal myocardial perfusion, which was evaluated by thallium scintigraphy. In addition, all cases demonstrated apparently normal hepatic LCFA accumulation Thus, these findings suggested that CD36 acts as a major myocardial specific LCFA transporter in humans.

[Reticulated platelet determination: methodologies and applications for the evaluation of thrombocytopenic disorders].

Reticulated platelets retain some residual mRNA in their cytoplasm and are thought to be newly produced platelets. In recent years, it has been reported that the reticulated platelet count (RP) correlates well with platelet production. For that reason, the measurement of RP (%) is considered useful for analyses of platelet kinetics and differential diagnoses of thrombocytopenic disorders. However, certain technical difficulties exist because fluorochrome thiazole orange (TO), which is used for staining purposes, stains platelet granules nonspecifically, and so far, only a few reports have documented the study of precision staining techniques. We evaluated staining criteria precisely in an effort to solve the issue of nonspecific staining by TO, and concluded that the important points for effective staining were (1) fixation of platelets, (2) 1:8 dilution of TO (ReticCount), (3) incubation for 1 to 2 hours, and (4) the capture of platelets using anti-CD42b monoclonal antibody. We stained reticulated platelet samples by the above method and achieved intra-assay reproducibility of 3.4-5.1% RP (%) in normal subjects was 8.7 +/- 2.2%. It was significantly higher (23.6 +/- 13.3%) in patients with idiopathic thrombocytopenic purpura (ITP), and elevated in 87% of all evaluated ITP patients. Our method is sensitive, provides reproducible results, and can be effectively utilized for the analysis of platelet kinetics and differential diagnosis of thrombocytopenia.

A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3.

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells.

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.

[The sensitivity, specificity and predictive value of PAIgG, reticulated platelets, thrombopoietin levels, and platelet size for the differential diagnosis of thrombocytopenia].

We evaluated measurements of PAIgG, reticulated platelets (RP), plasma thrombopoietin (TPO) levels, and platelet size to determine whether these parameters were useful for the differential diagnosis of idiopathic thrombocytopenic purpura (ITP), aplastic anemia (AA), and hypoplastic thrombocytopenia (HypoT). The percentage of RP (%RP) in patients with ITP was significantly higher (25.2 +/- 11.0%, P < 0.001) than in normal subjects (7.9 +/- 2.8), and the sensitivity, specificity, and predictive value of %RP in diagnosing ITP were 82%, 95%, 96%, respectively. On the other hand, TPO levels in patients with AA and HypoT were significantly higher (355.5 +/- 218.7 pg/ml, P < 0.001, and 376.4 +/- 347.2, P < 0.001, respectively) than in normal subjects (36.7 +/- 23.0). The sensitivity, specificity, and predictive value of TPO in diagnosing AA and HypoT were 88%, 89% and 86%, respectively. We also sought to determine whether the simultaneous measurement of %RP and TPO improved their value in the differential diagnosis of ITP, AA, and HypoT. However, simultaneous measurement did not yield significant improvements in sensitivty, specificity, or predictive value. These results indicated that measurements of %RP will suffice for the diagnosis of ITP, and that measurements of TPO are adequate for the diagnosis of AA and HypoT.

Involvement of Na+/Ca2+ exchanger in inside-out signaling through the platelet integrin IIbbeta3.

The platelet integrin IIbbeta3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbbeta3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbbeta3 remain obscure. Employing a chymotrypsin-induced IIbbeta3 activation model, we previously proposed the hypothesis that Na+/Ca2 + exchanger (NCX) may be involved in inside-out signaling (Shiraga et al: Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3',4'-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbbeta3 activation. Moreover, these inhibitors abolished IIbbeta3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets "thrombasthenic". These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbbeta3.

Isolation of PSD-Zip45, a novel Homer/vesl family protein containing leucine zipper motifs, from rat brain.

Using monoclonal antibody against the 45 kDa postsynaptic density protein, we isolated a novel isoform of Homer/vesl. The NH2-terminal region containing a PDZ domain of this protein is identical to that of Homer/vesl, and the COOH-terminal region containing unique leucine zippers shows self-multimerization. We named this protein PSD-Zip45. In addition to specific binding of PSD-Zip45 mediated by a PDZ domain to the metabotropic glutamate receptors 1alpha or 5, the distribution of PSD-Zip45 transcripts is highly consistent with that of metabotropic glutamate receptor transcripts. The PSD-Zip45 is, therefore, the first candidate as receptor anchoring proteins containing leucine zipper motifs in the central nervous system.