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Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Since its discovery as an essential component of the contractile machinery of striated muscle, the ability of purified actin to increase the viscosity of its aqueous solutions has been the object of many studies. Biochemical and biophysical studies soon established that the chemical stimuli, usually addition of monovalent or divalent salts, that increased viscosity did so coincident with promoting the polymerization of globular (G-) actin into filamentous polymers (F-actin). Defining how the properties of the individual filaments and their interactions with each other account for the macroscopic mechanical properties of F-actin solutions is an active area of research, with several fundamental questions remaining unanswered.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.

The specificity of cellular effects of lysolipid phosphate (LLP) growth factors is determined by binding to endothelial differentiation gene-encoded G protein-coupled receptors (EDG Rs), which transduce diverse proliferative and effector signals. The primary determinants of cellular responses to LLPs are the generative and biodegradative events, which establish steady-state concentrations of each LLP at cell surfaces, and the relative frequency of expression of each EDG R. There are major differences among types of cells in the net effective generation of the LLPs, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), and in their profile of expression of EDG Rs. The less well characterized secondary determinants of cellular specificity of LLPs are high-affinity binding proteins with carrier and cell-presentation functions, cell-selective regulators of expression of EDG Rs, and cellular factors that govern coupling of EDG Rs to G protein transductional pathways. The roles of components of the LLP-EDG R system in normal physiology and disease processes will be definitively elucidated only after development of animal models with biologically meaningful alterations in genes encoding EDG Rs and the discovery of potent and selective pharmacological probes.

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively, Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets, Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

Publisher Summary This chapter traces the development in the understanding of phagocytosis. Phagocytosis has a broad and a narrow definition. The former encompasses the spectrum of activities undertaken by a class of cells of the hematopoietic system in animal physiology related to tissue remodeling, immunity and inflammation. The latter is the attribute of particle engulfment that unifies this class of otherwise very diverse cells. Ilya Metchnikoff carried phagocytosis from its narrow to its broad conception. Moreover, the force of Metchnikoff's theory concerning the role of phagocytes in immunity and inflammation and the charm of the story by which he came to develop his theory have been so influential as to create the widespread belief that he discovered the narrower definition too, the fundamental cellular activity of phagocytosis. Metchnikoff cited the observation of an increased phagocytosis by leukocytes of microorganisms in immunized animals as evidence for his theory. But here he was on sorer ground, since the role of immunization in phagocytosis was later shown to be indirect, and since his adversaries marshaled evidence that infectious diseases prevailed in instances where there was brisk phagocytosis. Interestingly, some found Metchnikoff's ideas about battles between microbes and purposeful phagocytes too anthropomorphic and attacked him on this ground.

I have edited Current Opinion in Hematology for the past decade and am increasingly pleased with its high quality. This excellence is entirely due to the efforts of the Section Editors in their selection of outstanding topics and of distinguished authors to write about them. The willingness of the editors and authors to sacrifice the time and effort to produce such wonderful articles is a tribute to the journal’s value, reflected in its high impact factor.

This study investigates possible sources for the variance of more than two orders of magnitude in the published values for the shear moduli of purified actin filaments. Two types of forced oscillatory rheometers used in some of our previous work agree within a factor of three for identical samples. Polymers assembled in EGTA and Mg2+ from fresh, gel-filtered ATP-actin at 1 mg/ml typically have an elastic storage modulus (G') of approximately 1 Pa at a deformation frequency of 0.1-1 Hz. G' is slightly higher when actin is polymerized in KCl with Ca2+ and Mg2+. Gel filtration removes minor contaminants from actin but has little effect on G' for most preparations of actin from acetone powder. Storage of actin monomers without frequent changes of buffer containing fresh ATP and dithiothreitol can result in changes that increase the G' of filaments by more than a factor of 10. Frozen storage can preserve the properties of monomeric actin, but care is necessary to prevent protein denaturation or aggregation due to freezing or thawing.

Rac, a member of the rho family of GTPases, when activated transmits signals leading to actin-based membrane ruffling in fibroblasts. Compared with wild-type fibroblasts, gelsolin null (Gsn-) dermal fibroblasts have a markedly reduced ruffling response to serum or EGF stimulation, which signal through rac. Bradykinin-induced filopodial formation, attributable to activation of cdc42, is similar in both cell types. Wild-type fibroblasts exhibit typical lamellipodial extension during translational locomotion, whereas Gsn- cells move 50% slower using structures resembling filopodia. Multiple Gsn- tissues as well as Gsn- fibroblasts overexpress rac, but not cdc42 or rho, 5-fold. Re-expression of gelsolin in Gsn- fibroblasts by stable transfection or adenovirus reverts the ruffling response, translational motility and rac expression to normal. Rac migrates to the cell membrane following EGF stimulation in both cell types. Gelsolin is an essential effector of rac-mediated actin dynamics, acting downstream of rac recruitment to the membrane.

SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c-Ha-ras oncogene-transformed NIH/3T3 cells (EJ-NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ-NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A.Kuzumaki, N. (1993) Oncogene 8, 2531-2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild-type gelsolin in Escherichia coli, purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin-filament-severing activity and increased nucleating activity compared with wild-type gelsolin in vitro. Furthermore, compared to wild-type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 more strongly than wild-type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1-S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin-binding domains are in segments 1, 2, and 4-6, and that the region around the residue 321 may contain a phosphoinositol-lipid-binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras-transformed cells.

Cells crawl in response to external stimuli by extending and remodeling peripheral elastic lamellae in the direction of locomotion. The remodeling requires vectorial assembly of actin subunits into linear polymers at the lamella's leading edge and the crosslinking of the filaments by bifunctional gelation proteins. The disassembly of the crosslinked filaments into short fragments or monomeric subunits away from the leading edge supplies components for the actin assembly reactions that drive protrusion. Cellular proteins that respond to lipid and ionic signals elicited by sensory cues escort actin through this cycle in which filaments are assembled, crosslinked, and disassembled. One class of myosin molecules may contribute to crawling by guiding sensory receptors to the cell surface, and another class may contribute by imposing contractile forces on actin networks in the lamellae.

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

The motility of many cell types proceeds sporadically, by a sequence of propulsive and contractile movements. These movements appear to be controlled by proteins acting upon the actin cytoskeletal network which induce gel-sol transformations in specific regions of the cytoplasm. However, the forces that actually drive these cytoplasmic motions remain obscure; indeed there may be several force generating systems which dominate different types of motile events. For example, directed locomotion may involve the same direct mechanochemical coupling as occurs within muscles, in conjunction with other forces such as membrane bending, gel swelling and elasticity, or osmotic and hydrostatic pressures. In this chapter we will review the elastic properties of cytoskeletal protein networks. It is these networks that provide elastic resistance to cell deformation, and whose rearrangement may allow directed motion in response to externally or internally generated forces.

Networks of the biopolymer actin, cross-linked by the compliant protein filamin, form soft gels. They can, however, withstand large shear stresses due to their pronounced nonlinear elastic behavior. The nonlinear elasticity can be controlled by varying the number of cross-links per actin filament. We propose and test a model of rigid filaments decorated by multiple flexible linkers that is in quantitative agreement with experiment. This allows us to estimate loads on individual cross-links, which we find to be less than 10 pN. © 2009 The American Physical Society.

Thomas Stossel argues that restrictions on doctors’ and academics’ interaction with commercial companies are damaging research, but Kirby Lee believes it is a price worth paying to maintain public trust

Publisher Summary Actin assembles reversibly into linear polymers, and a large number of actin-binding proteins regulate this assembly and influence the architecture of actin filaments to produce a wide variety of three-dimensional structures. Actin and this family of actin-binding proteins constitute “the actin system.” Actin tends to concentrate in the periphery of cells in general and is, therefore, close to the plasma membrane, the shape of which it presumably influences and where it in turn can be regulated by signals generated by the interaction of membrane receptors with extracellular ligands. Although spectrin, a rodlike protein that assembles into tetramers, constitutes the principal mass of protein, in protein network that laminates the membrane of the red blood cell, actin oligomers (along with other proteins) link the spectrin molecules and therefore this cytoskeleton can be considered to be a member of the actin system. Molecules that bear names, such as fodrin and terminal web (TW) 260/240, contain epitopes immunoreactive with spectrin and resemble erythroid spectrin in size and shape. These properties as well as the presence in nonerythroid cells of proteins resembling ankyrin (the link between erythrocyte spectrin and band 3) in addition to other erythroid protein analogs, have led researchers to search for a “membrane skeleton” analogous to the spectrin–actin shell in eukaryotic cells.

The revolution in medicine and technology over the past few decades is largely the result of partnerships--or a "harmony of interests"--between private companies and entrepreneurial scientists and clinicians. Regulations to prevent conflicts of interest by restricting medical education, medical research, expert advisory functions, or researcher ownership of inventions may have the unintended consequence of slowing medical progress.

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet survival is unclear. We investigated the importance of sialic acid for platelet clearance using mice deficient in the ST3GalIV sialyltransferase gene (ST3GalIV−/− mice) and identified a novel clearance mechanism previously unrecognized for platelets. ST3GalIV catalyzes the addition of sialic acid onto exposed galactose residues of cell surface glycoproteins. ST3GalIV−/− mice have increased platelet surface galactose exposure, a 70% reduction in platelet count, and prolonged bleeding times. We report that ST3GalIV−/− platelets transfused into wild-type C57BL/6J mice exhibit markedly reduced recoveries and shortened survivals respectively compared to littermate wild-type platelets. Infusion of asialofetuin, an antagonist of the asialoglycoprotein-receptor (ASGPR) restored platelet recovery time and initial circulation of ST3GalIV−/− platelets to normal values. Immunohistochemical studies of organ specimens harvested shortly after transfusion of biotin-labelled platelets demonstrated the predominant clearance of ST3GalIV−/− platelets by the liver Kupffer cells and, unexpectedly, hepatocytes. Megakaryocytes cultured from ST3GalIV−/− mice produced proplatelets normally compared to megakaryocytes generated from wild-type littermates, indicating that the thrombocytopenia in the ST3GalIV−/− mice is not due to reduced platelet production. Comparison of ST3GalIV+/+ and ST3GalIV−/− platelet surface receptor expression as evidenced by flow cytometry and preliminary in vitro activation studies did not reveal any significant differences in the two genotypes. We conclude that the absence of terminal sialic acid residues on platelet surfaces exposes galactose residues to the lectin domain of ASGPR on both hepatocytes and liver Kupffer cells, resulting in platelet clearance from the circulation.

We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (>8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.

One pathway that mediates the rapid removal of transfused platelets exposed to cold for short periods (≤ 2 h) is the clustering of the von-Willebrand factor (VWF) receptor complex. Clustered VWF receptors are recognized by the αMβ2 integrin on hepatic macrophages, which, through its lectin domain, binds exposed βN-acetylglucosamine (βGlcNAc) residues on the clustered GPIbα subunits (Cell112: 87, 2003). Coverage of the exposed βGlcNAc residues by galactosylation prevents clearance of platelets from the circulation. We have now investigated detailed changes of the VWF complex on platelets during short (≤ 2 h) and prolonged refrigeration (≥ 48 h). The VWF receptor structure in chilled and rewarmed platelets was probed by flow cytometry using a panel of mAbs directed towards the GPIbα N-terminal (N–T) and GPIX (FITC-AN51 (aa36–59), PE-Cy5-HIP1 (aa59–81), FITC-SZ2 (aa268–282), and PE-GPIX mAb). Binding of the anti-GPIbα mAbs HIP1 and SZ2 as well as the anti-GPIX was diminished 40–50 % following 0.5 h of cooling when compared with room temperature (RT) platelets. The anti -GPIbα N-T mAb AN51 bound similarly to cooled and RT platelets. In contrast, the binding of all anti-VWF complex mAbs was decreased by 50–60 % after refrigeration of platelets in plasma for 48 h when compared to fresh RT platelets. Immunoblots demonstrated that loss of binding did not result from GPIbα shedding. VWF and fibrinogen binding were also increased by ~ 5 % or ~ 20 %, respectively, and P-selectin exposure increased by ~ 30 % in 48 h refrigerated platelets compared to the RT platelets stored for 48 h as determined by flow cytometry. These results suggest that the conformation of the VWF receptor is altered by long term refrigeration which leads to VWF binding and partial platelet activation. Loss of antibody binding is not simply due to GPIbα clustering as incubation of platelets with 5 μM latrunculin A for 1 h, which induces profound GPIbα clustering, increased binding by ~ 30 % compared to untreated platelets, but did not alter the diminished mAb binding to platelets induced by chilling. Refrigerated (≤ 14 days) platelets still adhere well to collagen-coated surfaces under flow (1000 seconds−1 for 2 min) demonstrating that VWF receptor function remains intact. We conclude that refrigeration of platelets in plasma for ≥ 48 h induces both conformational changes and clustering of the VWF complex, to prime it for activation by inducing VWF binding.

We recently presented evidence for the presence of a β4 galactosyl transferase (β4GalT) on the surface of platelets that catalyzes the coupling of galactose in a β1,4 linkage to exposed N-acetylglucosamine residues on the N-linked glycans of GP1ba and that this reaction improved the circulation of chilled murine platelets. Since an externally-disposed glycosyltransferase activity contradicts the dogma that such enzymes reside solely within the Golgi apparatus, we verified it by demonstrating that intact platelets glycosylate 2.8 μm beads coated with the acceptor substrate GlcNAc-β-R. We then explored the origin of this enzyme in megakaryocytes and its mechanism of delivery to platelets. We report that Golgi marker GM130 as well as a YFP tagged Golgi marker construct based on the polypeptide transferase GalNAc-T2 expressed in cultured mouse megakaryocytes transports from the cell body of to nascent platelets in discrete packets. In immature megakaryocytes, the Golgi organizes, as expected, into perinuclear arrays. However, once proplatelet extension begins, the perinuclear apparatus disassembles, and Golgi-associated proteins transport through the proplatelets in vesicular/granular structures. Labeling with granule markers reveals correspondence of Golgi markers only with dense granules. These findings suggest that Golgi membrane becomes packaged into dense granules, which deliver Golgi-associated glycosyltransferases into the nascent platelets and from there to the platelet surface.

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform named plasma gelsolin (pGSN). Relatively high (250 mg /L) blood concentrations of pGSN decrease in response to trauma, major surgery, sepsis, burns, ionizing radiation, and hyperoxia. Depletion of pGSN to a critical (~20%) level precedes and predicts complications of primary injuries such as lung permeability changes, ARDS, assisted ventilation and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. A proposed mechanism for pGSN’s protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. Because of its structural similarity we hypothesized that plasma gelsolin binds also to the potent lipid mediator platelet activating factor (PAF) and report here on the inhibition of PAF-induced cellular activation. Recombinant pGSN inhibited PAF-induced P-selectin up-regulation by human platelets as measured by flow cytometry. A ten- to 40-fold molar excess (0.5–20 μM) of pGSN over PAF inhibits P-selectin expression by 40 to 80%. The concentrations of plasma gelsolin used approximate the ~2–3 μM concentrations in plasma, and the molar excess of pGSN over PAF is probably greater in biological systems, where PAF has nanomolar affinity for its receptor. pGSN also inhibited PAF-induced superoxide anion (O2-) production (measured by chemiluminescence) of human neutrophils (PMN) in a concentration-dependent manner. The inhibition was up to 80% at a concentration of 10 μM (tenfold molar excess over PAF). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O2- generation by PMN. The inhibition was 65% at a 1:1 molar ratio (1 μM). In conclusion pGSN interferes with PAF-induced cellular activation in vitro, suggesting a mechanism for the protective role of plasma gelsolin that has been observed in vivo.

Over 30 years ago, Jamison and Barber proposed that externally disposed glycosyl transferase (GT) activity mediates platelet adhesion and other functions. Subsequent work ruled out ecto-GT activity in nucleated cells and established the Golgi as the site of such enzymes, although no further studies examined platelets. We recently reported a platelet-associated galactosyl transferase (β4Gal-T) catalyzing galactose coupling in a β1,4 linkage to exposed N-acetylglucosamine (GlcNAc) residues on N-linked glycans of the GPIbα subunit of the von Willebrand factor receptor complex. This activity, also present in plasma, mediates galactose transfer to GPIbα following addition of UDP-galactose (UDP-Gal) alone. Since galactosylation of GPIbα prevents clearance of chilled platelets by liver macrophages after transfusion, sufficiently active endogenous endogenous β4Gal-T could facilitate platelet cold storage after the mere addition of substrate UDP-Gal (Science301: 1531–1534, 2003). We now give evidence that human platelets contain a highly active (0.05 nmol/min) plasma membrane-associated ecto-β4Gal-T with properties of an isoform designated as type 1 (β4Gal-T1). A specific β4Gal-T1 antibody immunoprecipitates β4Gal-T1 activity from platelet lysates and the inhibition profile activity by α-lactalbumin and benzyl-β-D-GlcNAc are characteristic of β4Gal-T1. β4Gal-T1 has a transmembrane domain, and platelet β4Gal-T, although releasable by non-ionic detergent, remains cell- and membrane associated despite extensive washing. The platelet enzyme incorporates UDP-[C14]Gal into a single polypeptide with the apparent Mr of GPIbα, identifying this protein as its unique endogenous target. Nevertheless, washed platelets are as active as recombinant β4Gal-T1 in galactosylating exogenous substrates, benzyl-β-D-GlcNAc and ovalbumin. In conclusion, we have revived the concept of an ecto-glycosyl transferase on blood platelets and demonstrated that platelet β4Gal-T is probably sufficiently active to accommodate reliable enzymatic conversion of platelets by simple addition of UDP-Gal.

Cells, Gels and the Engines of Life A New, Unifying Approach to Cell Function. Gerald H. Pollack. Ebner, Seattle, 2001. 319 pp. Paper, $27.95. ISBN 0-9626895-2-1. Treating cells as polymer gels and stressing the physical chemistry of water and solutes, Pollack offers an accessible, non-mathematical challenge to mainstream approaches to cell biology.

To determine the national origins of high-quality clinical research we looked at research articles published during the past decade in three leading general clinical-research journals, the New England Journal of Medicine, the Journal of Clinical Investigation, and the Lancet, and in a specialty journal, Blood. We found that the proportion of non-U.S. papers published annually in these journals increased between two- and almost threefold, irrespective of whether the total number published per year rose (the Journal of Clinical Investigation and Blood), fell (the Lancet), or remained constant (the New England Journal of Medicine). Most non-U.S. research published in these periodicals originated in Western Europe or Japan. The limited available data on papers sent to the journals revealed a decline in the number of U.S. papers submitted in recent years (the New England Journal of Medicine) or a slower rate of increase relative to that of non-U.S. submissions (Blood), indicating that the increase in the number of non-U.S. papers published reflects an increase in the amount of high-quality research originating abroad as compared with the amount originating in the United States. The explanation for this phenomenon is unclear, but it coincides with the slowed growth of funding from the National Institutes of Health for U.S. clinical research.

Sera from 15 patients with antineutrophil antibodies against peripheral blood neutrophils were incubated with normal bone marrow, and the reaction with marrow precursors was determined. Serum from controls did not deposit IgG on either peripheral blood or marrow myeloid cells. Serum from patients with moderate immune neutropenia with myeloid hyperplasia deposited IgG on neutrophils, bands and metamyelocytes, but not on earlier precursors. Serum from one patient with severe neutropenia and myeloid hypoplasia deposited IgG on myelocytes and promyelocytes as well as more mature cells. Serum from a patient with antineutrophil antibodies which impaired chemotaxis but who did not have neutropenia deposited IgG only on neutrophils and occasional bands. Immune neutropenia may become increasingly severe as antibodies become directed against earlier precursors.

A large proportion of actin in extracts of human polymorphonuclear leukocytes is unpolymerized in the presence of 0.1 M KCl, a condition expected to promote maximal assembly of purified actin. Raising of the KCl concentration increases the extent of actin polymerization in leukocyte extracts, and, at KCl concentrations greater than or equal to 0.3 M, the polymer concentration is maximal and approaches that of purified muscle actin in 0.1 M KCl solution. After removal of the actin polymers from leukocyte extracts made 0.6 M in KCl, the supernatant fluid contains an activity which inhibits the extent of assembly of purified skeletal muscle actin in 0.1 M KCl solution. We have purified this activity by ion exchange and gel filtration chromatography. The purified inhibitor contains polypeptides of 65,000 and 62,000 daltons in a molar ratio of 1:2 as determined by polyacrylamide gel electrophoresis in dodecyl sulfate. The Stokes radius of 32 A, and S20,w of 4.8 of the native inhibitor suggest that the purified protein is a mixture of monomers of slightly different molecular weight. Substoichiometric concentrations of the inhibitor rapidly prevent the assembly of purified actin in 0.1 m KCl solution in a concentration-dependent manner, and the reduction in final viscosity remains constant over time. Raising the KCl concentration reverses the inhibition, as observed for actin polymerization in unfractionated extracts. The findings are consistent with the conclusion that this inhibitor protein is responsible for the large proportion of unpolymerized actin in extracts of polymorphonuclear leukocytes.

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.