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We investigated the occurrence of the unusual phenomenon of hemoglobin polymerization in a 10-year survey of 47 species of fishes. Similar to human sickle cell disease, hemoglobin polymers in fish red blood cells can cause distortion or sickling under low oxygen and low pH. We sampled fish from three geographic areas, including the east and west coasts of the Atlantic Ocean and the Gulf of Mexico. Fifteen species spanning five orders and nine families exhibited hemoglobin polymerization in vitro, with a majority in or related to Gadiformes, as well as species within Notocanthi-formes, Perciformes, and Scorpianiformes. Atlantic cod, Gadus morhua, also showed the trait in vivo. Light and transmission electron microscopy confirmed the presence of hemoglobin polymers at the cellular level, but the morphology of hemoglobin polymers and rates of polymerization varied across species. Hemoglobin polymerization in red blood cells in vitro was pH dependent and reversible. For two species, G. morhua and Opsanus tau, >60% and >40% of all red blood cells contained hemoglobin polymers at pH 7.6, while 100% and 90% of red blood cells polymerized at pH 6.96, respectively. In both species, recovery of 60%-70% of red blood cells occurred within 45 minutes when pH increased from 6.96 to 7.99. From these results we conclude that hemoglobin polymerization is present in a broad range of fish taxa occupying wide biogeo-graphical ranges and habitats and that it is oxygen and pH sensitive. The physiology and adaptive significance of hemoglobin polymerization in fishes remain unclear, but as oceans and coastal environments become more hypoxic and hypercapnic, this trait may have the potential to affect fish survival., Introduction Hemoglobins (Hbs) for transport of [O.sub.2] in the blood remain the most intensively studied proteins for their function and structural variations. Globins or globin-like genes have been described in [...]

We provide an overview of our studies on cadmium and the actin cytoskeleton in mesangial cells, from earlier work on the effects of [Cd.sup.2+] on actin polymerization in vivo and in vitro, to a role of disruption or stabilization of the cytoskeleton in apoptosis and apoptosis-like death. More recent studies implicate cadmium-dependent association of gelsolin and the [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMK-II) with actin filaments in cytoskeletal effects. We also present previously unpublished data concerning cadmium and the disruption of focal adhesions. The work encompasses studies on rat, mouse, and human mesangial cells. The major conclusions are that [Cd.sup.2+] acts independently of direct effects on cellular [Ca.sup.2+] levels to nevertheless activate [Ca.sup.2+]-dependent proteins that shift the actin polymerization-depolymerization in favour of depolymerization. Cadmium-dependent translocation of CaMK-IIS, gelsolin, and a 50 kDa gelsolin cleavage fragment to the filamentous (F-)actin cytoskeleton appear to be involved. An intact filamentous actin cytoskeleton is required to initiate apoptotic and apoptotic-like death, but F-actin depolymerization is an eventual result. Key words: cadmium toxicity, actin polymerization, cytoskeleton, calcium/calmodulin-dependent protein kinase-II, gelsolin, apoptosis, reactive oxygen species, mesangial cell. Nous presentons un survol des etudes que nous avons realisees sur le cadmium et le cytosquelette d'actine dans les cellules mesangiales, de nos premiers travaux sur les effets du [Cd.sup.2+] sur la polymerisation de l'actine in vivo et in vitro, jusqu'a un role de la desintegration ou la stabilisation du cytosquelette dans la mort par apoptose ou de type apoptose. Des etudes plus recentes impliquent l'association dependante du cadmium de la gelsoline et de la proteine kinase dependante du [Ca.sup.2+]/calmoduline II (CaMKII) aux filaments d'actine dans les effets du cytosquelette. Nous presentons aussi des donnees anterieures non publiees qui concernent le cadmium et l'interruption des adhesions focales. Ces travaux comprennent des etudes sur les cellules mesangiales du rat, de la souris et de l'humain. Les principales conclusions sont que le [Cd.sup.2+] agit independamment des effets directs sur les niveaux de [Ca.sup.2+] cellulaire pour activer malgre tout des proteines dependantes du [Ca.sup.2+] qui deplacent l'equilibre polymerisation-depolymerisation en faveur de la depolymerisation. La translocation dependante du cadmium de la CaMK-IIS,de la gelsoline et d'un fragment de 50 kDa issu du clivage de la gelsoline vers le cytosquelette d'actine filamenteuse (F-) semble impliquee. Un cytosquelette intact d'actine filamenteuse est requis pour initier la mort par apoptose ou de type apoptose, mais la depolymerisation de la F-actine est un resultat a long terme. [Traduit par la Redaction] Mots-cles : toxicite du cadmium, polymerisation de l'actine, cytosquelette, proteine kinase dependante du [Ca.sup.2+]/calmoduline II, gelsoline, apoptose, espece reactive d'oxygene, cellule mesangiale., Cadmium toxicity and reactive oxygen species Cadmium is an occupationally and environmentally important toxic element that is present in the air, soil, sediment, and water. Non-occupational exposure is mainly from [...]

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/ vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with [alpha]-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with [alpha]-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC. Ena/VASP; EVH1; phosphorylation; profilin doi:10.1152/ajpcell.00431.2009

Am J Physiol Cell Physiol 298: C182-C190, 2010. First published October 14, 2009; doi:10.1152/ajpcell.00326.2009.--The phenomenon of posttetanic potentiation, in which a single submaximal contraction or series of submaximal contractions strengthens a subsequent contraction, has been observed in both skeletal and cardiac muscle. In this study, we describe a similar phenomenon in swine carotid arterial smooth muscle. We find that a submaximal [K.sup.+] depolarization increases the force generation of a subsequent maximal [K.sup.+] depolarization; we term this 'force augmentation.' Force augmentation was not associated with a significant increase in crossbridge phosphorylation or shortening velocity during the maximal [K.sup.+] depolarization, suggesting that the augmented force was not caused by higher crossbridge phosphorylation or crossbridge cycling rates. We found that the characteristics of the tissue before the maximal [K.sup.+] depolarization predicted the degree of force augmentation. Specifically, measures of stimulated actin polymerization (higher prior Yl18 paxillin phosphorylation, higher prior F-actin, and transition to a more solid rheology evidenced by lower noise temperature, hysteresivity, and phase angle) predicted the subsequent force augmentation. Increased prior contraction alone did not induce force augmentation since readdition of [Ca.sup.2+] to [Ca.sup.2+]-depleted tissues induced a partial contraction that was not associated with changes in noise temperature or with subsequent force augmentation. These data suggest that stimulated actin polymerization may produce a substrate for increased crossbridge mediated force, a process we observe as force augmentation. phase angle; noise temperature; paxillin; rheology; vascular smooth muscle

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.09.021 Byline: Frauke Drees (1), Sabine Pokutta (1)(2), Soichiro Yamada (1), W. James Nelson (1), William I. Weis (1)(2) Abstract: Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that [alpha]-catenin links the adhesion protein E-cadherin through [beta]-catenin to the actin cytoskeleton, in the accompanying paper we report that [alpha]-catenin does not bind simultaneously to both E-cadherin-[beta]-catenin and actin filaments. Here we demonstrate that [alpha]-catenin exists as a monomer or a homodimer with different binding properties. Monomeric [alpha]-catenin binds more strongly to E-cadherin-[beta]-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that [alpha]-catenin is an allosteric protein. Significantly, [alpha]-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for [alpha]-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion. Author Affiliation: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA (2) Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA Article History: Received 31 March 2005; Revised 28 July 2005; Accepted 12 September 2005 Article Note: (miscellaneous) Published: December 1, 2005

The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelia[ cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. We describe a novel role for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. We also examined other actin regulators. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein [beta] enhance abl phenotypes.

During the 5 s of the acrosome reaction of Limulus polyphemus sperm, a 60-[micro]m-long bundle of scruin-decorated actin filaments straightens from a coiled conformation and extends from the cell. To identify the motive force for this movement, we examined the possible sources of chemical and mechanical energy and show that the coil releases ~[10.sup.-13] J of stored mechanical strain energy, whereas chemical energy derived from calcium binding is ~[10.sup.-15] J. These measurements indicate that the coiled actin bundle extends by a spring-based mechanism, which is distinctly different from the better known polymerization or myosin-driven processes, and that calcium initiates but does not power the reaction.

In vitro polymerization of the essential bacterial cell division protein FtsZ, in the presence of GTP, is rapid and transient due to its efficient binding and hydrolysis of GTP. In contrast, the in vivo polymeric FtsZ structure which drives cell division - the Z-ring - is present in cells for extended periods of time whilst undergoing constant turnover of FtsZ. It is demonstrated that dynamic polymerization of Escherichia coli FtsZ in vitro is sensitive to the ratio of GTP to GDP concentration. Increase of GDP concentration in the presence of a constant GTP concentration reduces both the duration of FtsZ polymerization and the initial light-scattering maximum which occurs upon addition of GTP. It is also demonstrated that by use of a GTP-regeneration system, polymers of FtsZ can be maintained in a steady state for up to 85 min, while preserving their dynamic properties. The authors therefore present the use of a GTP-regeneration system for FtsZ polymerization as an assay more representative of the in vivo situation, where FtsZ polymers are subject to a constant, relatively high GTP to GDP ratio.

The bacterial peptidoglycan consists of glycan chains of repeating [beta]-1,4-linked N-acetylglucosaminyl--N-acetylmuramyi units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The [beta]-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters [K.sub.m] and [k.sub.cat]/[K.sub.m] were measured at 40.6 [micro]M [+ or -] 15.5) and 1 x [10.sup.-3] [M.sup.-1] [s.sup.-1], respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40[degrees]C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20[degrees]C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the [tau] and [gamma] subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40[degrees]C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34[degrees]C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39[degrees]C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 30[degrees]C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38[degrees]C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.

Research has been conducted on the atrane-like six-coordinate (RO) (sub)2 TaL, where R=Me or Et and L= tris(2-oxy-3,5-dimethylbenzyl)amine, complexes with three six-membered rings. The synthesis and characterization of these complexes have been carried out and the details are presented.

Research has been conducted on the C4b-binding protein, a complement activation inhibitor. The ability of the protein's alpha-chains to polymerize has been tested and the results are reported.

Research has been conducted on the models with the growing radical chains formed from diallylamine and diallylammonium monomers. The results of the radical cyclization reactions of these models investigated via the use of the density functional theory are presented.

We show that in solutions of human hemoglobin (Hb)--oxy- and deoxy-Hb A or S--of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40[degrees]C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.

Research has been conducted on the hydrogen chloride. The computational results of the hydrogen chloride elimination from Cl (sub)3 GaNH (sub)3 and intermediates' gas-phase polymerization are presented.

Research has been conducted on the xanthan, an exopolysaccharide produced by a plant-pathogenic bacterium. The purification and characterization of Bacillus sp. strain GL1 enzyme have been carried out in investigating the degradation pathway of this xanthan and the nature of alpha-mannosidase in bacteria and the details are presented.

Research has been conducted on the profilin and profilin-actin complex interactions with actin filament barbed end. The role of profilin in the nonmuscle beta,gamma actin elongation has been investigated and the details are reported.

Research has been conducted on the cyclic esters' ring-opening polymerization. The structural characterization of the iron(III)-alkoxide complexes and the comparative studies of the compounds' behavior have been carried out and are described.

The authors review the publications on hydrated amphiphiles forming different phases as a function of the molecular structure, temperature, concentration and pressure. The topics of interest include the methods used for hydrated amphiphile polymerization in bilayer membranes and in nonlamellar phases and the characteristics of the resultant polymeric materials and polymers.

All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASp [DELTA] VCA) in [WAS.sup.-/-] mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor [alpha][beta] expression and a consequent failure to generate single-positive [CD4.sup.+] and [CD8.sup.+] T cells. These latter defects, which are not observed in [WAS.sup.-/-] mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Atp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Atp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.

Research has been conducted on the stabilizing cap at microtubule ends. Results indicate that this cap consists of the single layer tubulin GDP-P (sub)i subunits and that the P (sub)i release mechanism destabilizes GDP-tubulin core.

Results demonstrate that hydrolysis of magnesium bound ATP actin is three times faster as shown by mechanical quenched-flow technique. Hydrolysis rates of the point end and the barbed end are similar with no effect on the latter by saturating concentration of profilin.

Research has been conducted on the 2,2'-bis(2-indenyl)biphenyl derivatives. The applicable route to these derivatives has been developed and the results are presented.

Research has been conducted on the group 4 dimethylmetallocenes, the catalyst precursors for the methylmetallocenium/borate catalyst systems for olefin polymerization. The reaction of the pi-ligand with the 2-fold excess of MeLi and then MtCl (sub)4 where Mt is Ti, Zr, Hf carried out in preparing the bisindenyldimethylmetallocenes has been investigated and the details are reported.

The authors review the publications on the structure and function of the Arp2/3 complex. The topics of interest include the crystal structure, the cryo-EM structure and the reconstruction of the complex from the subunits.

The authors review the publications on the in vitro enzyme-catalyzed organic reactions. The topics of interest include the advances in enzymatic polymerizations and the nature of the catalyst enzymes.

Research has been conducted on the living radical polymerization. The points of interest include the metal-catalyzed living radical polymerization, transition-metal catalysts and monomers.

The authors review the publications on the radical polymerization. The topics of interest include nitroxide-mediated living free radical polymerization, strategies for polymeric structure and macromolecular architecture control and the design of nitroxides with improved performance.

The author reviews publications on living radical polymerization subfield. The topics of interest include the aspects important for the persistent radical effect operation in living polymerization.

The authors review the publications on free-radical polymerization. The topics of interest include tacticity of polymerization, reactivity ratios of monomers and polymerization rate controlled by the Lewis acid addition.

Research has been conducted on the 16a'-homo-leurosidine. The enantioselective synthesis of this compound and its extension to the synthesis of its C-10' epimer 16a'-homo-vinblastine is described.

Research has been conducted on the 5a'-homo-vinblastine and its C-20' methyl congener. The synthesis of these compounds has been carried out and the results show that the compounds do no allow isolation of atropisomers.

Research has been conducted on the scaffolding proteins required for the viruses to direct capsid assembly. A model of the scaffolding protein that selectively stabilizes the on-pathway growing intermediates in case of bacteriophage P22 has been investigated.