D-半乳糖诱导大鼠脑损伤的糖基化机制

目的D-半乳糖（D-galactose）诱导大鼠体内不同糖基化水平,研究其脑损伤发生的机理。方法采用不同剂量D-半乳糖[150、75、37．5mg／（kg·d）]分别腹腔注射（ip）处理大鼠8周,诱导糖基化状态和脑损伤。采用硫代巴比妥酸（TBA）比色法测定糖化血红蛋白,硝基四氮唑蓝（NBT）比色法测定血清果糖胺;按文献方法分别测定血红细胞醛糖还原酶活性和晚期糖基化终末产物（AGEs）含量及脑组织中AGEs含量,羟胺法和比色法分别测定SOD和GSH-Px活性,硫代巴比妥酸法测定MDA含量;以Fura-2／AM作为钙荧光指示剂,双波长荧光分光光度法检测脑海马神经细胞胞质[Ca^2＋]i的变化;透射电镜观察脑海马神经细胞线粒体的变化。结果D-半乳糖处理8周后,大鼠血红细胞醛糖还原酶活性升高,糖化产物形成增多;脑组织中AGEs及脑细胞胞质[Ca^2＋]i含量明显升高,SOD及GSH-Px活性下降,MDA含量升高（P〈0．01,P〈0．05）,海马神经细胞线粒体出现病理性改变。结论D-半乳糖通过诱导体内蛋白糖基化和脑组织AGEs大量生成,降低抗氧化能力及胞质[Ca^2＋]i超负荷等,导致脑细胞损伤。     

Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

Alterations in the balance of cytoskeleton as well as energetic proteins are involved in the cardiac remodeling occurring in dilated cardiomyopathy (DCM). We used two-dimensional DIGE proteomics as a discovery approach to identify key molecular changes taking place in a temporally controlled model of DCM triggered by cardiomyocyte-specific serum response factor (SRF) knock-out in mice. We identified muscle creatine kinase (MCK) as the primary down-regulated protein followed by α-actin and α-tropomyosin down-regulation leading to a decrease of polymerized F-actin. The early response to these defects was an increase in the amount of desmin intermediate filaments and phosphorylation of the αB-crystallin chaperone. We found that αB-crystallin and desmin progressively lose their striated pattern and accumulate at the intercalated disk and the sarcolemma, respectively. We further show that desmin is a preferential target of advanced glycation end products (AGE) in mouse and human DCM. Inhibition of CK in cultured cardiomyocytes is sufficient to recapitulate both the actin depolymerization defect and the modification of desmin by AGE. Treatment with either cytochalasin D or glyoxal, a cellular AGE, indicated that both actin depolymerization and AGE contribute to desmin disorganization. Heat shock-induced phosphorylation of αB-crystallin provides a transient protection of desmin against glyoxal in a p38 MAPK-dependent manner. Our results show that the strong down-regulation of MCK activity contributes to F-actin instability and induces post-translational modification of αB-crystallin and desmin. Our results suggest that AGE may play an important role in DCM because they alter the organization of desmin filaments that normally support stress response and mitochondrial functions in cardiomyocytes.     

Actin microfilaments in fungi

The cytoskeletal protein actin is among the most abundant proteins in nature. It is almost ubiquitous, occurring in all eukaryotes and in an ancestral form in prokaryotes. Actin monomers can polymerise to form microfilaments, structures that play a critical role in a number of fundamental cell processes in fungi such as morphogenesis, cytokinesis and the movement of organelles. Microfilaments are extremely dynamic structures and can be rapidly modified through their interactions with a number of actin binding proteins (ABPs). The purpose of the following review is to introduce actin and microfilaments in fungi to a general mycological audience and to provide a basic framework from which further study is possible.     

Effect of Experimental Diabetes on the Catecholamine Metabolism in Rat Brain

The levels of epinephrine, norepinephrine, and dopamine and the activities of tyrosine hydroxylase and monoamine oxidase were estimated in four regions of rat brain during alloxan-induced hyperglycemia and insulin-induced hypoglycemia. Catecholamine levels were estimated by HPLC, and the insulin levels were quantified by radioimmunoassay. The results demonstrated significant increases in the activities of the metabolizing enzymes and levels of catecholamines during experimental conditions. The levels of catecholamines were highest in the cerebral hemispheres, the region associated with high activities of the metabolizing enzymes. Insulin-induced hypoglycemia caused a decrease in the activities of the metabolizing enzymes followed by their recovery within 2 h.     

Role of zinc metallothionein-3 (ZnMt3) in epidermal growth factor (EGF)-induced c-Abl protein activation and actin polymerization in cultured astrocytes

Recent evidence indicates that zinc plays a major role in neurochemistry. Of the many zinc-binding proteins, metallothionein-3 (Mt3) is regarded as one of the major regulators of cellular zinc in the brain. However, biological functions of Mt3 are not yet well characterized. Recently, we found that lysosomal dysfunction in metallothionein-3 (Mt3)-null astrocytes involves down-regulation of c-Abl. In this study, we investigated the role of Mt3 in c-Abl activation and actin polymerization in cultured astrocytes following treatment with epidermal growth factor (EGF). Compared with wild-type (WT) astrocytes, Mt3-null cells exhibited a substantial reduction in the activation of c-Abl upon treatment with EGF. Consistent with previous studies, activation of c-Abl by EGF induced dissociation of c-Abl from F-actin. Mt3 added to astrocytic cell lysates bound F-actin, augmented F-actin polymerization, and promoted the dissociation of c-Abl from F-actin, suggesting a possible role for Mt3 in this process. Conversely, Mt3-deficient astrocytes showed significantly reduced dissociation of c-Abl from F-actin following EGF treatment. Experiments using various peptide fragments of Mt3 showed that a fragment containing the N-terminal TCPCP motif (peptide 1) is sufficient for this effect. Removal of zinc from Mt3 or pep1 with tetrakis(2-pyridylmethyl)ethylenediamine abrogated the effect of Mt3 on the association of c-Abl and F-actin, indicating that zinc binding is necessary for this action. These results suggest that ZnMt3 in cultured astrocytes may be a normal component of c-Abl activation in EGF receptor signaling. Hence, modulation of Mt3 levels or distribution may prove to be a useful strategy for controlling cytoskeletal mobilization following EGF stimulation in brain cells.     

Malaria Parasite Actin Polymerization and Filament Structure

A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and “zebra-striped” appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter- and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility.     

Posttranslational Processing of Brain Actin

Abstract: Two short-lived isoforms of actin, named δ- and ε-actin, have been detected in brain extracts from rats labeled in vivo with [³⁵S]methionine. These two molecular species have PI values slightly more basic than β- and γ-actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ- and ε-actin, synthesized in vivo , can be converted in vitro into β- and γ-actin. This posttranslational processing of δ- and ε-actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH₂-terminal end, is involved in the transformation of these proteins into β- and γ-actin.     

Mono-ADP-Ribosylation in Brain: Purification and Characterization of ADP-Ribosyltransferases Affecting Actin from Rat Brain

Four ADP-ribosyltransferases that acted on non-muscle actin were purified more than 3,000-fold from rat brain extract. The molecular weights of these brain ADP-ribosyltransferases were 66,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on TSK gel G3000SW. The Km values for NAD were approximately 20 microM. These enzymes were not inhibited by thymidine or nicotinamide, but were inhibited by ADP and ADP-ribose. Two soluble ADP-ribosylation factors purified from rat brain had different effects on the four ADP-ribosyltransferases during the ADP-ribosylation of non-muscle actin. These ADP-ribosyltransferases modified not only actin but also the stimulatory guanine nucleotide-binding protein of adenylate cyclase, Gs, and another guanine nucleotide-binding protein in brain, Go. These findings suggest that the four brain ADP-ribosyltransferases are concerned with nerve functions in the central nervous system.     

Changes in Monoamine Oxidase Activity in Rat Brain During Alloxan Diabetes

Abstract: The effect of alloxan diabetes on the activity of monoamine oxidase was studied in three regions of the rat brain at various time intervals after the onset of diabetes. It was observed that monoamine oxidase activity was decreased at early time intervals after diabetes, followed by a recovery in all three regions of the brain. A reversal of the effect was observed with insulin administration to the diabetic rats.     

Catching the WAVEs of Plant Actin Regulation

Plants, as all other eukaryotic organisms, depend on a dynamic actin cytoskeleton for proper function and development. Actin dynamics is a complex process, regulated by a number of actin-binding proteins and large multiprotein complexes like ARP2/3 and WAVE. The ARP2/3 complex is recognized as a nucleator of actin filaments, and it generates a highly branched network of interlaced microfilaments. Results from multiple organisms show that ARP2/3 activity is regulated through multiple pathways. Recent results from plants point to a signaling pathway leading from the small GTPase RAC/ROP through a protein complex containing the ARP2/3-activating protein WAVE. This signaling pathway appears to be evolutionarily conserved. Support for this regulatory mechanism comes from studies of mutations in genes encoding subunits of the putative ARP2/3 complex and the WAVE complex in Arabidopsis. Several such mutants have defects of actin filament organization, leading to a conspicuous “distorted” trichome phenotype. Multiple growth and developmental phenotypes reported for napp/gnarled/atnap, pirp/pirogi/atpir, and distorted3 mutants reveal that these WAVE proteins are also required for a wider variety of cellular functions in addition to regulating trichome cell growth. These results have implications for the current view on cell morphogenesis in plants.     

Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca²⁺ homeostasis.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.     

Drebrin-induced Stabilization of Actin Filaments

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (α_sk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated α_sk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH₂ terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γ_cyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γ_cyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γ_cyto-actin (but not α_sk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-α_sk-actin or TM-β/γ_cyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γ_cyto-actin (but not α_sk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.     

A 130-kDa Protein 4.1B Regulates Cell Adhesion,Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization

Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998