Respiratory Complexes III and IV Are Not Essential for the Assembly/Stability of Complex I in Fungi

The functional relevance of respiratory supercomplexes in various eukaryotes including mammals, plants, and fungi is hitherto poorly elucidated. However, substantial evidence indicates as a major role the assembly and/or stabilization of mammalian complex I by supercomplex formation with complexes III and IV. Here, we demonstrate by using native electrophoresis that the long-lived Podospora anserina mutant Cyc1-1, respiring exclusively via the alternative oxidase (AOX), lacks an assembled complex III and possesses complex I partially assembled with complex IV into a supercomplex. This resembles the situation in complex-IV-deficient mutants displaying a corresponding phenotype but possessing I-III supercomplexes instead, suggesting that either complex III or complex IV is in a redundant manner necessary for assembly/stabilization of complex I as previously shown in mammals. To corroborate this notion, we constructed the double mutant Cyc1-1,Cox5::ble. Surprisingly, this mutant lacking both complexes III and IV is viable and essentially a phenocopy of mutant Cyc1-1 including the reversal of the phenotype towards wild-type-like characteristics by the several-fold overexpression of the AOX in mutant Cyc1-1,Cox5::ble(Gpd-Aox). Fungal specific features (not found in mammals) that must be responsible for assembly/stabilization of fungal complex I when complexes III and IV are absent, such as the presence of the AOX and complex I dimerization, are addressed and discussed. These intriguing results unequivocally prove that complexes III and IV are dispensable for assembly/stability of complex I in fungi contrary to the situation in mammals, thus highlighting the imperative to unravel the biogenesis of complex I as well as the true supramolecular organization of the respiratory chain and its functional significance in a variety of model eukaryotes. In summary, we present the first obligatorily aerobic eukaryote with an artificial, simultaneous lack of the respiratory complexes III and IV.     

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9–10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.     

A Thermodynamic Model of Microtubule Assembly and Disassembly

Microtubules are self-assembling polymers whose dynamics are essential for thenormal function of cellular processes including chromosome separation andcytokinesis. Therefore understanding what factors effect microtubule growth isfundamental to our understanding of the control of microtubule based processes.An important factor that determines the status of a microtubule, whether it isgrowing or shrinking, is the length of the GTP tubulin microtubule cap. Here, wederive a Monte Carlo model of the assembly and disassembly of microtubules. Weuse thermodynamic laws to reduce the number of parameters of our model and, inparticular, we take into account the contribution of water to the entropy of thesystem. We fit all parameters of the model from published experimental datausing the GTP tubulin dimer attachment rate and the lateral and longitudinalbinding energies of GTP and GDP tubulin dimers at both ends. Also we calculateand incorporate the GTP hydrolysis rate. We have applied our model and can mimicpublished experimental data, which formerly suggested a single layer GTP tubulindimer microtubule cap, to show that these data demonstrate that the GTP cap canfluctuate and can be several microns long.     

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.     

Synthesis,Spectral and Antibacterial Studies of Binuclear Titanium(IV) / Zirconium(IV) Complexes of Piperazine Dithiosemicarbazones

The reactions of mono(cyclopentadienyl)titanium(IV) trichloride and bis(cyclopentadienyl)titanium(IV)/ zirconium(IV) dichloride with a new class of dithiosemicarbazone, derived by condensing piperazine dithiosemicarbazide with benzaldehyde (L₁H₂), 2-chlorobenzaldehyde (L₂H₂), 4-nitrobenzaldehyde (L₃H₂) or salicylaldehyde (L₄H₄) have been studied and different types of binuclear products, viz. [{CpTiCl₂}₂L], [{Cp₂MCl}₂L], ((L=L₁, L₂ or L₃), [{CpTiCI}₂L₄] and [{Cp₂M}₂L₄] (M=Yi or Zr), have been isolated. Tentative structures are proposed for these complexes based upon elemental analyses, electrical conductance, magnetic moment and spectral (electronic, IR, ¹H and ¹³C NMR) data. Attempts have been made to establish a correlation between antibacterial activity and the structures of the products.     

JAK2 and SHP2 Reciprocally Regulate Tyrosine Phosphorylation and Stability of Proapoptotic Protein ASK1

Previously we have shown that tyrosine 718 of ASK1 when phosphorylated is critical for SOCS1 binding and SOCS1-mediated degradation of ASK1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are unknown. In this study, we identified JAK2 and SHP2 as a Tyr-718-specific kinase and phosphatase, respectively. Interferon-γ (IFN-γ) induced degradation of ASK1 in normal but not in SOCS1-KO endothelial cells (EC). IFN-γ-induced tyrosine phosphorylation of ASK1 at Tyr-718 was blocked by a JAK2-specific inhibitor. IFN-γ enhanced the association between JAK2 and ASK1, and the ASK1-JAK2 complex was labile and was stabilized by the proteasomal inhibitor MG132. Furthermore, JAK2, but not JAK1, directly bound to and phosphorylated ASK1 at Tyr-718, leading to an enhanced association of ASK1 with SOCS1 and subsequent ASK1 degradation. Next, we showed that overexpression of the SH2-containing protein-tyrosine phosphatase-2 (SHP2) augmented, whereas a phosphatase-inactive mutant of SHP2 inhibited, TNF-induced ASK1 dephosphorylation. SHP2 associated with ASK1 in response to tumor necrosis factor in EC. An SHP-2 substrate-trapping mutant formed a complex with tyrosine-phosphorylated ASK1, suggesting that ASK1 is a direct SHP2 substrate. Moreover, SHP2 wild type, but not a catalytically inactive mutant, dissociated SOCS1 from ASK1. IFN-γ-induced ASK1 Tyr(P)-718 was enhanced in mouse EC deficient in SHP2 (SHP2-KO). In contrast, tumor necrosis factor-induced dephosphorylation of ASK1 at Tyr(P)-718 and activation of ASK1-JNK signaling, as well as EC apoptosis, are significantly reduced in SHP2-KO EC. Our data suggest that JAK2-SOCS1 and SHP2 reciprocally regulate ASK1 phosphorylation and stability in response to cytokines.Myocardial infarction due to atherosclerosis of coronary arteries remains the leading cause of death in the United States. It has become clear that increases in inflammatory mediators represent a common pathogenic mechanism for atherosclerosis (1). The vascular cell that normally limits the inflammatory and atherosclerotic process is the EC.³ Proinflammatory stimuli induce EC dysfunction, which is characterized by an enhanced sensitivity of vascular cells to proinflammatory and proapoptotic stimuli. Studies from our laboratory and others have demonstrated that ASK1 (apoptosis signal-regulating kinase-1), a member of MAP3K family (2, 3), is an effector of inflammation in EC (4–8). Almost all inflammatory stimuli such as tumor necrosis factor-α (TNF), interleukin-1 (IL-1), and reactive oxygen species activate ASK1. Activated ASK1 subsequently recruits and activates its downstream target MAP2Ks (MKK3/7 and MKK4/7), which in turn activate MAPKs (JNK and p38). Studies from ASK1-deficient mice have also linked ASK1 to cardiovascular pathogenesis. ASK1 deletion in mice attenuated angiotensin II-induced cardiac hypertrophy and remodeling. Neointimal formation due to proliferation of smooth muscle cells in a cuff injury model is also attenuated by ASK1 deletion in mice (9, 10).Although the linkage of ASK1 to inflammation is very strong, the mechanism by which inflammatory stimuli, including TNF, activate ASK1 is not fully understood. The identification of proteins associated with ASK1 and their regulation on ASK1 have provided some insights into the mechanism for ASK1 activation. ASK1 is a 170-kDa protein that is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. One important regulatory mechanism of ASK1 activity is its Ser/Thr phosphorylation and dephosphorylation by kinases and phosphatases. ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site and inhibits ASK1 activity (11, 12). TNF activates ASK1 in part by dissociating these cellular inhibitors from ASK1 (4, 7). Recently, we have identified PP2A as a phosphatase in TNF-induced dephosphorylation of ASK1 Ser(P)-967 (13). In addition to the 14-3-3-binding site, Ser(P)-967, ASK1 is phosphorylated at Ser-83 by Akt, leading to inhibition of ASK1 activity. In contrast, autophosphorylation of ASK1 at Thr-838 leads to oligomerization and activation (14). Phosphorylation of Thr-845 can be negatively regulated by the phosphatase PP5 (15). Similarly, we found that the ASK1 autophosphorylation at Thr-813 and Thr-842 also positively regulates ASK1 signaling (16).In contrast to Ser/Thr phosphorylation, regulation of ASK1 by tyrosine phosphorylation is less well understood. We have recently shown that ASK1 is phosphorylated at Tyr-718, and this phosphorylation is critical for the binding to suppressor of cytokine signaling-1 (SOCS1), a subunit of ubiquitin ligase responsible for ASK1 degradation (17). Tyrosine phosphorylation of ASK1 is up-regulated in response to growth factors and cytokines such as IFN-γ, whereas this phosphorylation can be down-regulated by TNF treatment, resulting in ASK1 dissociation from SOCS1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are not known.The cytoplasmic tyrosine kinase, JAK2, autophosphorylates in response to growth factors and cytokines, including IFN-γ. JAK2 then activates cytokine receptors and other cytoplasmic proteins such as the STATs by phosphorylating their key tyrosine residue. The JAK/STAT pathway can be regulated by SH2-containing protein-tyrosine phosphatases such as SHP2 (18–20). SHP2 is ubiquitously expressed and composed of two SH2 domains on the N-terminal and C-terminal protein-tyrosine phosphatase (PTP) domain. The SH2 domain of SHP2 mediates the association with phosphotyrosine-containing proteins present on activated receptors as well as on activated JAKs and STATs; this association triggers activation of the tyrosine phosphatase domain and subsequent dephosphorylation of substrates. SHP2 signals downstream of receptor tyrosine kinases and cytokine receptors, and in most cases it serves to positively transduce signals from these receptors. In other instances SHP2 has been shown to exhibit inhibitory signaling properties by negatively regulating the JAK-STAT pathway (19).In this study, we demonstrate that the IFN-γ-activated kinase JAK2 and TNF-activated SHP2 are the tyrosine kinase and phosphatase for Tyr-718 on ASK1, respectively. The actions of both JAK2 and SHP2 affect protein turnover of ASK1 and thus regulate ASK1/JNK-dependent proinflammatory and proapoptotic pathways in EC.     

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.     

c-Type Cytochrome Assembly in Saccharomyces cerevisiae: A Key Residue for Apocytochrome c1/Lyase Interaction

The electron transport chains in the membranes of bacteria and organelles generate proton-motive force essential for ATP production. The c-type cytochromes, defined by the covalent attachment of heme to a CXXCH motif, are key electron carriers in these energy-transducing membranes. In mitochondria, cytochromes c and c₁ are assembled by the cytochrome c heme lyases (CCHL and CC₁HL) and by Cyc2p, a putative redox protein. A cytochrome c₁ mutant with a CAPCH heme-binding site instead of the wild-type CAACH is strictly dependent upon Cyc2p for assembly. In this context, we found that overexpression of CC₁HL, as well as mutations of the proline in the CAPCH site to H, L, S, or T residues, can bypass the absence of Cyc2p. The P mutation was postulated to shift the CXXCH motif to an oxidized form, which must be reduced in a Cyc2p-dependent reaction before heme ligation. However, measurement of the redox midpoint potential of apocytochrome c₁ indicates that neither the P nor the T residues impact the thermodynamic propensity of the CXXCH motif to occur in a disulfide vs. dithiol form. We show instead that the identity of the second intervening residue in the CXXCH motif is key in determining the CCHL-dependent vs. CC₁HL-dependent assembly of holocytochrome c₁. We also provide evidence that Cyc2p is dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁.THE c-type cytochromes, also referred to as cytochrome c, represent a universal class of heme-containing proteins that function as electron carriers in the energy-transducing pathways of bacteria, plastids, and mitochondria (Thöny-Meyer 1997; Nakamoto et al. 2000; Bonnard et al. 2010). Because cytochromes c carry a heme covalently attached to a CXXCH motif, they constitute an attractive object of study to address the question of cofactor protein assembly. The biochemical requirements for cytochrome c assembly were deduced from in vivo and in vitro studies, and the conclusion is that both apocytochromes c and heme are transported independently across at least one biological membrane and maintained as reduced prior to catalysis of the heme attachment reaction (Allen et al. 2003; Hamel et al. 2009; Kranz et al. 2009; Sanders et al. 2010). Bacterial cytochromes c are assembled in the periplasmic space, a compartment where cysteine pairs in proteins form disulfide bonds in reactions catalyzed by dedicated enzymes (Inaba 2009; Kadokura and Beckwith 2010). The current thinking holds that a c-type apocytochrome is a substrate of the disulfide bond-forming pathway, which introduces an intramolecular disulfide between the two cysteines of the CXXCH sequence (Allen et al. 2003; Sanders et al. 2010). This disulfide needs to be reduced to a dithiol to provide free sulfhydryls for the heme ligation. Consistent with this view is the fact that groups of specific oxido-reductases that constitute a transmembrane dithiol-disulfide relay from the cytosol to the periplasmic space have been shown to function as c-type cytochrome assembly factors (Allen et al. 2003; Kadokura et al. 2003; Mapller and Hederstedt 2006; Sanders et al. 2010). The proposal that the components of this pathway control the in vivo redox status of the CXXCH sulfhydryls has been inferred from the presence of motifs in their protein sequences that are consistent with a function in redox chemistry and also from the demonstration that their recombinant forms participate in dithiol–disulfide exchange reactions (Monika et al. 1997; Setterdahl et al. 2000). Moreover, the ability of exogenous thiol compounds to bypass the lack of these factors in vivo substantiates the view that the redox components have a disulfide-reducing activity in the pathway (e.g., Sambongi and Ferguson 1994; Fabianek et al. 1998; Beckett et al. 2000; Deshmukh et al. 2000; Bardischewsky and Friedrich 2001; Erlendsson and Hederstedt 2002; Erlendsson et al. 2003; Feissner et al. 2005; Turkarslan et al. 2008).While the role of these pathways is well established in bacteria, much less is known about the components that catalyze thiol/disulfide chemistry in the mitochondrial intermembrane space (IMS), which is topologically equivalent to the bacterial periplasm. By analogy with the bacterial pathways, the participation of redox-active factors that catalyze thiol formation is expected, as the mitochondrial IMS houses two c-type cytochromes, the soluble cytochrome c and the membrane-bound cytochrome c₁, both of which function in respiration. In fungi, heme attachment to apocytochromes c and c₁ is dependent upon the IMS resident cytochrome c and c₁ heme lyases, CCHL and CC₁HL, although the exact role of these lyases in the assembly process is still unclear (Dumont et al. 1987; Zollner et al. 1992). Conversion of apocytochrome to holocytochrome c depends only on CCHL, while apocytochrome c₁ can be acted upon by both CCHL and CC₁HL (Matner and Sherman 1982; Dumont et al. 1987; Stuart et al. 1990; Zollner et al. 1992; Bernard et al. 2003). In animals, apoforms of cytochromes c and c₁ are assembled by a unique heme lyase, HCCS, which carries both the CCHL and CC₁HL activities (Prakash et al. 2002; Schwarz and Cox 2002; Bernard et al. 2003).Cyc2p, a component first described as a mitochondrial biogenesis factor in yeast (Matner and Sherman 1982; Dumont et al. 1993; Pearce et al. 1998; Sanchez et al. 2001), was recently rediscovered in the context of cytochrome c₁ maturation (Bernard et al. 2003). Cyc2p is located at the mitochondrial inner membrane with its C-terminal domain containing a non-covalently bound FAD exposed to the IMS (Bernard et al. 2005). A redox function for Cyc2p is likely based on the finding that a recombinant form of the molecule exhibits a NAD(P)H-dependent reductase activity (Bernard et al. 2005). However, as Cyc2p activity is not essential for the maturation process, a functional redundancy was postulated based on the fact that a cyc2-null mutant still assembles holoforms of cytochromes c and c₁ (Bernard et al. 2005). The absolute requirement of Cyc2p was revealed via genetic analysis of the cyc2-null cyt1-34 combination that displays a synthetic respiratory-deficient phenotype with loss of holocytochrome c₁ assembly (Bernard et al. 2005). The cyt1-34 mutation maps to the gene encoding cytochrome c₁ and results in a CAPCH heme-binding site replacing the wild-type CAACH site (Bernard et al. 2005). The synthetic interaction is specific for the cyt1-34 allele carrying the A-to-P mutation and is not observed in a cyc2-null cyt1-48 strain carrying an A-to-D mutation at the heme-binding site of apocytochrome c₁ (Bernard et al. 2005). The fact that Cyc2p becomes essential when the cytochrome c₁ heme-binding site carries an A-to-P mutation suggests that the CXXCH motif could be the target of Cyc2p action in vivo. One possible interpretation for this observation is that the P residue alters the reactivity of the cysteinyl thiols to redox chemistry so that the apocytochrome c₁ CAPCH heme-binding site occurs in an oxidized (disulfide) form, which must be reduced in a Cyc2p-dependent reaction before heme attachment can occur.In this article, we have undertaken a genetic approach to elucidate this pathway and searched for suppressors that alleviate the respiratory deficiency of the cyc2-null cyt1-34 strain. Either overexpression of CC₁HL or replacement of the P mutation in the heme-binding site by H, L, S, or T residues restore the assembly of holocytochrome c₁. In vitro measurement of redox potential of apoforms of CA(A/P/T)CH cytochrome c₁ indicates that there is no change in the thermodynamic stability of the disulfide at the CXXCH motif that could account for the Cyc2p-dependent assembly of cytochrome c₁. Genetic studies reveal that the replacement of the second A residue at the CAACH motif by H, L, P, S, and T residues is key in determining the conversion of apocytochrome c₁ to its corresponding holoform via the CCHL and/or CC₁HL-dependent pathway. We also demonstrate that Cyc2p is a component dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁. We propose that the CAPCH cytochrome c₁ is strictly dependent upon CCHL and Cyc2p for its assembly but becomes a substrate of CC₁HL upon overexpression of CC₁HL or in the presence of H, L, S, or T mutations.     

The Structure of the Catalytic Domain of a Plant Cellulose Synthase and Its Assembly into Dimers

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.     

The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse

The mTORC1 pathway is required for both the terminal muscle differentiation andhypertrophy by controlling the mammalian translational machinery viaphosphorylation of S6K1 and 4E-BP1. mTOR and S6K1 are connected by interactingwith the eIF3 initiation complex. The regulatory subunit eIF3f plays a majorrole in muscle hypertrophy and is a key target that accounts for MAFbx functionduring atrophy. Here we present evidence that in MAFbx-induced atrophy thedegradation of eIF3f suppresses S6K1 activation by mTOR, whereas an eIF3f mutantinsensitive to MAFbx polyubiquitination maintained persistent phosphorylation ofS6K1 and rpS6. During terminal muscle differentiation a conserved TOS motif ineIF3f connects mTOR/raptor complex, which phosphorylates S6K1 and regulatesdownstream effectors of mTOR and Cap-dependent translation initiation. ThuseIF3f plays a major role for proper activity of mTORC1 to regulate skeletalmuscle size.     

Influence of Urea,Hydroxyurea, and Thiourea on Meloidogyne javanica and Infected Excised Tomato Roots in Culture

Urea (U), hydroxyurea (HU), and thiourea (TU), in various concentrations, were added to chemically defined plant tissue culture medium on which Meloidogyne javanica was reared on excised tomato roots. Concentrations as low as 3 ppm HU or 12 ppm TU inhibited nematode maturation by 70-90% 4 weeks after inoculation, and the coenocytes in the parasitized tissue were poorly developed. Gall weight was also inhibited by 50% in cultures treated with 3 and 6 ppm HU. However, exposing juveniles of M. javanica and Tylenchulus semipenetrans or juveniles and adults of Pratylenchus thornei to increasing concentrations of HU or TU, up to 100 ppm, was not lethal. These two urea derivatives still inhibited nematode maturation when the infected region of the root was not in direct contact with the chemicals. Therefore, we suggest that these urea derivatives inhibit nematode development by affecting the plant metabolism essential to coenocyte formation, an occurrence similar to the hypersensitive reaction in a naturally resistant plant.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Current status of Paragonimus and paragonimiasis in Ecuador

A review of national and international publications on paragonimiasis in Ecuador,epidemiological records from the Ministry of Public Health and unpublished researchdata was conducted to summarise the current status of the parasite/disease. Thepurpose of the review is to educate physicians, policy-makers and health providers onthe status of the disease and to stimulate scientific investigators to conductfurther research. Paragonimiasis was first diagnosed in Ecuador 94 years ago and itis endemic to both tropical and subtropical regions in 19 of 24 provinces in thePacific Coast and Amazon regions. Paragonimus mexicanus is the onlyknown species in the country, with the mollusc Aroapyrgus colombiensisand the crabs Moreirocarcinus emarginatus,Hypolobocera chilensis and Hypolobocera aequatorialisbeing the primary and secondary intermediate hosts, respectively. Recentstudies found P. mexicanus metacercariae in Trichodactylusfaxoni crabs of the northern Amazon. Chronic pulmonary paragonimiasis iscommonly misdiagnosed and treated as tuberculosis and although studies havedemonstrated the efficacy of praziquantel and triclabendazole for the treatment ofhuman infections, neither drug is available in Ecuador. Official data recorded from1978-2007 indicate an annual incidence of 85.5 cases throughout the 19 provinces,with an estimated 17.2% of the population at risk of infection. There are no currentdata on the incidence/prevalence of infection, nor is there a national controlprogramme.     

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.     

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c 551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA cyt c ₅₅₁), and Hydrogenobacter thermophilus cytochrome c ₅₅₂ (HT cyt c ₅₅₂), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c ₅₅₂ forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c ₅₅₁ also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c ₅₅₁ were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.