Oligomerization of a peptide was attempted in a flow reactor that simulated a submarine hydrothermal system. When fluid containing glycine repeatedly circulated through the hot and cold regions in the reactor, oligopeptides were made from glycine. When divalent ions (such as copper ions) were added under acidic conditions, oligoglycine was elongated up to hexaglycine. This observation suggests that prebiotic monomers could have oligomerized in the vicinity of submarine hydrothermal vents on primitive Earth.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolinhomologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actinrich Triton X-100-insoluble pellets following platelet aggregation induced by thrombin receptor-activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actinand actin-related protein 2 and 3 (Arp2/3)-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/ Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets. IntroductionReorganization of cortical actin filaments plays a critical role in cell movement and pattern formation. 1,2 WASP (Wiskott-Aldrich syndrome protein), N-WASP, and WAVE (WASP family Verprolinhomologous protein, or Scar), called WASP family proteins, are likely to regulate cortical actin filament reorganization in response to extracellular stimuli, although not all of these proteins have been linked to actin polymerization. 1,2 Each of these proteins has a verprolin-homology (V) domain, cofilin-homology (C) domain, and an acidic (A) region at the C-terminus, which are necessary to enhance the intrinsic actin polymerization activity of the actinrelated protein 2 and 3 (Arp2/3) complex. [1][2][3][4][5][6][7][8][9] Platelets are essential for normal hemostasis and have an unusually high actin content (0.5 mM). 10,11 In suspension, platelets are rapidly transformed from disks to spheres with spiny protrusions upon stimulation by agonists such as adenosine diphosphate, collagen, and thrombin. [10][11][12][13][14][15] This "shape change" is inhibited by cytochalasins, which prevent actin polymerization. [10][11][12][13][14][15] Platelets also adhere onto various surfaces and rapidly spread, a process that is also inhibited by cytochalasins. Thus, actin polymerization is pivotal in the rapid morphologic transformation of platelets in the initial phase of hemostasis. Among the WASP and WAVE family proteins, platelets express WASP, which becomes strongly tyrosine phosphorylated upon stimulation by collagen or following crosslinking of CD32, a low-affinity Fc receptor for immunoglobulin G (IgG). 16 However, despite the...

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein.In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.CrkL is a Src homology (SH)2 1 and SH3 adapter (1-3). Through its SH2 domain, CrkL binds to focal adhesions proteins like paxillin and Cas (1-3). CrkL also binds to a Rapspecific guanine nucleotide exchange factor, C3G, through its N-terminal SH3 domain and, thus, conveys C3G to focal adhesions. C3G activates a small GTPase Rap1 and regulates cell adhesion and spreading, indicating that the CrkL-C3G complex is a critical component of focal adhesions (4). We previously reported that CrkL is present in human platelets, and that it is an adapter for WASP, syk, or phosphorylated STAT5 (5-7).On the other hand, ADP-ribosylation factors (Arfs) are also members of the Ras-related small GTPases and function in the regulation of membrane trafficking and actin cytoskeleton (8, 9). Similar to other GTPases, the activity of Arfs is regulated positively by GEFs and negatively by GTPase-activating proteins (GAPs). Recently, several Arf GAPs have been cloned and characterized and found to have phosphoinositide-dependent GAP activity toward Arfs (10). ASAP1 (also called DEF-1, for differentiation enhancing factor-1), the prototype of the phosphoinositide-dependent Arf GAP family, is a multidomain protein with pleckstrin homology, Arf GAP, ankyrin repeat, proline-rich region, and SH3 domains. ASAP1 binds to phosphatidylinositol (4, 5)P 2 through its PH domain and shows GAP activity toward Arf (11-13). In NIH3T3 cells, endogenous ASAP1 localizes in focal adhesions and, when overexpressed, ASAP1 affects cell spreading of NIH3T3 cells on fibronectin (14). Although the function of Arf in focal adhesions is not clear yet, the localization of ASAP1 and its effect on cell spreading suggests the importance of Arf signaling on the dynamics of focal adhesions.During our continual efforts to clarify the role of CrkL in the regulation of signal transduction, we found that the SH3 domain of CrkL binds to ASAP1. The data obtained from studies using platelets and COS7 cells overexpressing ASAP1 revealed that CrkL is a critical lynchpin between ASAP1 and focal adhesions. EXPERIMENTAL PROCEDURESBlo...

SummaryCD36 deficiency was studied with attention to the phenotypegenotype relationship. The diagnosis of CD36 deficiency was made when CD36 was negative on platelets (type II) or on both platelets and monocytes (type I). Among 827 apparently healthy Japanese volunteers, the type I and II deficiencies were found in 8 (1.0%) and 48 (5.8%), respectively. The T for C substitution at nt478 for Pro90Ser and the insertion of A at nt1159 constituted the major causes of type I and II deficiencies. The dinucleotide deletion at nt539 had a minor role. In two family studies, we found a previously unreported polymorphic site in the 5’-proximal flanking region and the 3’-untranslated region. Including these new polymorphisms, DNA sequence other than the three known mutations affecting CD36 expression was not observed in the CD36 gene, calling into question the previous hypothesis that a platelet-specific silent allele exists near or at the CD36 gene.

No abstract

Summary We have investigated the inhibitory effect on experimental or spontaneous lung metastases of polypeptides which contain repetitive structures of the Arg-Gly-Asp (RGD) or Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence derived from adhesion molecules, and studied their biological characterisation after administration. In the spontaneous metastasis model, multiple intravenous (i.v.) administrations of poly (RGD) and poly (YIGSR) resulted in a reduction of lung tumour colonies, although the monomer peptides, RGD or YIGSR, had no effect under these conditions. The treatment with poly(RGD) substantially prolonged the survival time for mice injected i.v. with B16-BL6 cells as compared to the treatment with RGD and random poly(R, G, D). Tumour cell adhesion to the fibronectin-substrates was remarkably inhibited by adding poly(RGD) freely in solution. Poly(RGD) was found to inhibit completely the ability of platelets to enhance tumour cell adhesion to fibronectin-substrate and tumour cell-elicited platelet aggregation in vitro, but poly(R, G, D) had no such effect. We also found that poly(RGD) led to a decrease in the arrest and retention of tumour cells after its co-injection with radiolabelled tumour cells and that the radiolabelled polypeptide can be at least decomposed into small fragments during circulation. Poly(RGD) was found to be still active in inhibiting experimental lung metastasis even when the contributions of NK cells or macrophages were removed from this system after pretreatment with anti-asialo GM1 serum, 2-chloroadenosine or carrageenan. The results indicate that the poly(RGD)-mediated inhibition of tumour metastasis may be due to the interference of the adhesive interaction of tumour cells with a specific site in the target organs. Derivatives of polypeptides which contain RGD and/or YIGSR sequences derived from cell adhesion proteins may thus provide a promising approach for the control and prevention of cancer metastasis.

Monoclonal anti CD36 antibodies capable of inhibiting platelet adhesion to collagen have not previously been identified. We have now prepared two groups of monoclonal antibodies. One group was prepared using, as immunogen, highly purified (99+%) CD36 prepared by a denaturing procedure. These antibodies (Mo series) reacted strongly with CD36 on protein blots but did not immunoprecipitate native CD36 from platelet lysates nor inhibit platelet adhesion to collagen. The second group of monoclonal antibodies (131 series) was prepared using CD36 purified to >95% by a non-denaturing procedure. These antibodies reacted with control platelets, but not Nak(a)-negative platelets which lack CD36, as measured by flow cytometry and immunoprecipitation. Three monoclonal antibodies of this latter group (131.4, 131.5 and 131.7) inhibited platelet adhesion to collagen in static systems under Mg2+ -independent conditions but had lit tle effect in the presence of Mg2+. 131.4 and 131.7 also inhibited adhesion to collagen using citrated whole blood in a parallel plate flow chamber at physiological shear rates (800s-1), whereas 131.5 was without effect. These are the first anti-CD36 monoclonal antibodies shown to be capable of inhibiting platelet adhesion to collagen and provide further evidence that CD36 plays a role in platelet-collagen interaction.

The mechanism underlying bacterial conjugation through protozoa was investigated. or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH67-vital stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. Kanamycin-resistant Escherichia coliIn this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.