Ultrastructural localization of photosystem I and II active sites in chloroplasts in situ]

Activities related to photosystem I and photosystem II are cytochemically localized at the electron microscope level by using suitable artificial electron donors and acceptors with, or without, factors uncoupling the light-induced electron flow between the two systems. Photo-oxidation of DAB, insensitive to DCMU, is linked to photosystem I activity. It occurs within the grana and stroma membranes of the chloroplasts. Photo-reduction of TC-NBT sensitive to DCMU, is a photosystem II mediated reaction. It is trapped within the grana membranes.     

Structure and function of type I copper in multicopper oxidases

The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordinating 1Met or the non-coordinating 1Phe/1Leu, and it exhibits spectral properties and an alkaline transition similar to those of the blue copper center in blue copper proteins. Since the type I copper center in multicopper oxidases is deeply buried inside the protein molecule, electron transfers to and from type I copper are performed through specific pathways: the hydrogen bond between an amino acid located at the substrate binding site and a His residue coordinating type I copper, and the His-Cys-His sequence connecting the type I copper center and the trinuclear copper center comprised of a type II copper and a pair of type III coppers. The intramolecular electron transfer rates can be tuned by mutating the fourth ligand of type I copper. Further, mutation at the Cys ligand gives a vacant type I copper center and traps the reaction intermediate during the four-electron reduction of dioxygen.     

Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129^(3.49) or E240^(6.30) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G_q and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.     

Nitric oxide: a radical molecule in quest of free radicals in proteins

A number of enzymes use an amino acid free radical cofactor. Tyrosyl and tryptophanyl radicals react with nitric oxide (NO) with an almost diffusion-limited rate. The catalytically competent tyrosyl radical in ribonucleotide reductase (RR) and prostaglandin H synthase (PGHS) recombines with NO in a radical-radical reaction. The unstable adduct formed can dissociate to regenerate the tyrosyl radical. However, upon prolonged incubation with NO, the diiron center of mouse RR leaks out, while the adduct is sucessively oxidized into an iminoxyl radical and a nitrotyrosine in PGHS. These data provide a plausible mechanism for the physiological inactivation of RR observed in various models, and may help in understanding the inhibition of PGHS reported in some cases. Reversible combination with NO is an intrinsic property of tyrosyl radicals, which also occurs with Y_D ^• and Y_Z ^• in photosystem II, where NO has been useful in the analysis of the oxygen-evolving complex.     

Serine residue 115 of MAPK-activated protein kinase MK5 is crucial for its PKA-regulated nuclear export and biological function

The mitogen-activated protein kinase-activated protein kinase-5 (MK5) resides predominantly in the nucleus of resting cells, but p38^MAPK, extracellular signal-regulated kinases-3 and -4 (ERK3 and ERK4), and protein kinase A (PKA) induce nucleocytoplasmic redistribution of MK5. The mechanism by which PKA causes nuclear export remains unsolved. In the study reported here we demonstrated that Ser-115 is an in vitro PKA phosphoacceptor site, and that PKA, but not p38^MAPK, ERK3 or ERK4, is unable to redistribute MK5 S115A to the cytoplasm. However, the phosphomimicking MK5 S115D mutant resides in the cytoplasm in untreated cells. While p38^MAPK, ERK3 and ERK4 fail to trigger nuclear export of the kinase dead T182A and K51E MK5 mutants, S115D/T182A and K51E/S115D mutants were able to enter the cytoplasm of resting cells. Finally, we demonstrated that mutations in Ser-115 affect the biological properties of MK5. Taken together, our results suggest that Ser-115 plays an essential role in PKA-regulated nuclear export of MK5, and that it also may regulate the biological functions of MK5.     

What’s new in the renin-angiotensin system?

The type 1 angiotensin receptor (AT(1)) activates an array of intracellular signalling pathways that control cell and tissue responses to the peptide hormone angiotensin II (AngII). The capacity of AT(1) receptors to initiate and maintain such signals has typically been explained on the basis of conventional heterotrimeric guanine nucleotide binding protein (G protein) activation, specifically G(q/11). Accumulating evidence from studies utilising a variety of AT(1) receptor mutants and AngII analogues indicates that some important downstream effects of AT(1) receptors are independent of classical G protein coupling. Importantly, AT(1) receptor-mediated endocytosis, tyrosine phosphorylation signalling and mitogen-activated protein kinase activation as well as transactivation of the epidermal growth factor receptor can occur in G(q/11)-uncoupled receptor mutants. These observations point to a functional partitioning of AT(1) receptor signals that permits separation of short-term AngII actions (e.g., vasoconstriction) from more extended events, such as pathological cell growth in heart and blood vessels, and may open up new avenues for selective antagonism.     

Transduction of the chemotactic cAMP signal across the plasma membrane ofDictyostelium cells

AggregatingDictyostelium cells secrete cAMP during cell aggregation. cAMP induces two fast responses, the production of more cAMP (relay) and directed cell locomotion (chemotaxis). Extracellular cAMP binds to G-protein-coupled receptors leading to the activation of second messenger pathways, including the activation of adenylyl cyclase, guanylyl cyclase, phospholipase C and the opening of plasma membrane Ca²⁺ channels. Many genes encoding these sensory transduction proteins have been cloned and null mutants of nearly all components have been characterized in detail. Undoubtedly, activation of adenylyl cyclase is the most complex, involving G-proteins, a soluble protein called CRAC and components of the MAP kinase pathway. Null mutants in this pathway do not aggregate, but can exhibit chemotaxis and develop normally when supplied with exogenous cAMP. The pathways leading to the activation of phospholipase C were identified, but unexpectedly, deletion of the phospholipase C gene has no effect on chemotaxis and development, nor on intracellular Ins(1,4,5)P₃ levels; the metabolism of this second messenger will be discussed in some detail. Activation of guanylyl cyclase is G-protein-dependent and essential for chemotaxis. Analysis of a collection of chemotactic mutants reveals that most mutants are defective in either the production or intracellular detection of cGMP, thereby placing this second messenger at the center of chemotactic signal transduction. Analysis of the cAMP-mediated opening of plasma membrane calcium channels in signal transduction mutants suggests that it has two components, one that depends on G-proteins and intracellular cGMP and one that is G-protein-independent.     

分枝杆菌噬菌体 D29的生物学特性研究及其抗耐药结核潜力的探讨

目的研究分枝杆菌噬菌体 D29的生物学特性,为 D29抗耐药结核治疗奠定基础.方法观察噬菌体电镜结构和噬菌斑形态;测定 D29最佳感染复数(MOI);一步生长实验;检测 pH值对 D29活力的影响;斑点法测定裂解谱;中和实验检测抗原性.结果 D29噬菌斑圆形透明,边界清楚;D29尾长129nm,最佳 MOI为10-4;D29感染宿主菌的潜伏期约为50min,裂解量为10;pH值对 D29存活率影响大,酸性环境不影响 D29裂解能力;D29能裂解分枝杆菌临床耐药株;D29K值为1069.50.结论 D29属于长尾噬菌体科(siphoviridae),裂解谱广,抗原性较高,具有抗耐药结核潜力     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3′ untranslated region (3′UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3′UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3′UTR target site inhibits translation directed by the HCV 5′UTR. Thus, the miR-122 target sites in the 3′-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.     

Iron-sulfur proteins: Recent developments in the field

Summary Iron-sulfur clusters in proteins are now recognized as among the main types of electron-transferring groups in biological systems, besides heme and flavins. Recent developments have brought forth a better understanding about the ways the protein environment modulates the potential of the cluster by placing the cluster in a more or less hydrophobic surrounding. Refinement in models, extensive studies on the kinetics of electron transfer (e.g. by measurement of the electronic spin lattice relaxation time) and the introduction of novel spectroscopic methods (EXAFS, magnetic CD and others) in the elucidation of structures in various systems are among the main developments. Other advances include EPR studies of the spatial orientation of Fe−S centers in complex membraneous systems (e.g. in mitochondria) and the recent elucidation of the nature of center X in photosystem I by M?ssbauer-spectroscopy. M?ssbauer studies have also been described on a number of Fe−S proteins (nitrogenase, aconitase, some ferredoxins, etc.) and revealed the existence of novel structures that enlarged the number of known basic units of Fe−S centers. These advances include: 1. the discovery of a novel non-heme Fe-protein (called desulforedoxin) of the rebredoxin type, 2. the elucidation of the nitrogenase Fe−S centers and the nitrogenase cofactor and 3. the discovery of a three-iron cluster in several enzymes and some ferredoxins. The latter 3-Fe cluster seems capable of being converted into a classical 4-Fe cluster under appropriate conditions, a phenomenon that plays a role in activation-deactivation of some enzymes (e.g. aconitase). It is now recognized that some iron-sulfur clusters may be involved in systems devoided of any oxydation-reduction reaction and may act as sensors of the surrounding redox potential, triggering the activation/deactivation of an enzyme (cf. e.g. aconitase).     

Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis

The D1 protein of Photosystem II (PSII), encoded by the psbA genes, is an indispensable component of oxygenic photosynthesis. Due to strongly oxidative chemistry of PSII water splitting, the D1 protein is prone to constant photodamage requiring its replacement, whereas most of the other PSII subunits remain ordinarily undamaged. In cyanobacteria, the D1 protein is encoded by a psbA gene family, whose members are differentially expressed according to environmental cues. Here, the regulation of the psbA gene expression is first discussed with emphasis on the model organisms Synechococcus sp. and Synechocystis sp. Then, a general classification of cyanobacterial D1 isoforms in various cyanobacterial species into D1_m, D1:1, D1:2, and D1′ forms depending on their expression pattern under acclimated growth conditions and upon stress is discussed, taking into consideration the phototolerance of different D1 forms and the expression conditions of respective members of the psbA gene family.     

Effects of cytochalasin D on the actin cytoskeleton: association of neoformed actin aggregates with proteins involved in signaling and endocytosis

Cytochalasin D (CD) has been extensively used for assessing the role of the actin cytoskeleton in different biological processes. However, effects of CD have not always been consistent and CD-treated cells have been found to contain irregular spots of F-actin. By transfecting MCF-7 cells with an actin-enhanced yellow fluorescent protein fusion protein we show that, in vivo, CD induces actin aggregation de novo, while simultaneously depolymerizing preexisting actin cytoskeletal components. We also show that CD-induced actin aggregates bind the F-actin-selective drug phalloidin and associate with proteins involved in cell signaling as well as with receptors and endosomal markers (active MAP kinases, paxillin, erbB2, transferrin, Rab-5), but not with clathrin, protein kinase A, protein tyrosine phosphatase 1B, or tubulin. Thus, CD induces new sites of actin aggregation that selectively associate with several important regulatory proteins. Failure of CD to interupt a biological process may therefore not prove that the process is independent of actin aggregation.     

The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum

Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum, several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium. Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.     

Effect of increasing concentrations of detergents on electron transfer in chloroplasts]

The effects of increasing concentrations of sodium cholate and deoxycholate, "Tween 80" and "Triton X 100" upon O2 evolution by intact or osmotically broken spinach chloroplasts were investigated polarographically. Except for cholate, low concentrations of any detergent increased considerably (up to four times) the rates of oxygen evolution by broken chloroplasts, without interrupting the electron flux between photosystem II and photosystem I.     

Preferences of transmembrane helices for cooperative amplification of Gαs and Gαq signaling of the thyrotropin receptor

The majority of constitutively activating mutations (CAMs) of the thyroid-stimulating hormone receptor display a partially activated receptor. Thus, full receptor activation requires a multiplex activation process. To define impacts of different transmembrane helices (TMHs) on cooperative signal transduction, we combined single CAMs in particular TMHs to double mutations and measured second messenger accumulation of the G_αs and the G_αq pathway. We observed a synergistic increase for basal activity of the G_αs pathway, for all characterized double mutants except for two combinations. Each double mutation, containing CAMs in TMH2, 6 and 7 showed the highest constitutive activities, suggesting that these helices contribute most to G_αs-mediated signaling. No single CAM revealed constitutive activity for the G_αq pathway. The double mutations with CAMs from TMH1, 2, 3 and 6 also exhibited increase for basal G_αq signaling. Our results suggest that TMH2, 6, 7 show selective preferences towards G_αs signaling, and TMH1, 2, 3, 6 for G_αq signaling.