Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.     

Distribution of neuraminidase and n-acetylneuraminate lyase activities among corynebacteria, mycobacteria, and nocardias

In Corynebacterium diphtheriae and closely related neuraminidase-producing corynebacteria, we have found an N-acetylneuraminate (NAN) lyase activity which cleaves NAN into N-acetyl-d-mannosamine and, presumably, pyruvate. In vitro, these lyases can be shown to synthesize NAN. A survey of representative corynebacteria, "plant pathogenic corynebacteria," mycobacteria, and nocardias revealed that only those corynebacteria closely related to C. diphtheriae exhibited both neuraminidase and NAN lyase activities.     

Ablation of connexin43 in smooth muscle cells of the mouse intestine: functional insights into physiology and morphology

Connexin43 (Cx43) gap-junction channels are highly abundant in intestinal smooth muscle but their functional impact has not been studied so far. Here, we have aimed to elucidate the functional role of Cx43 in the tunica muscularis of the mouse intestine in vivo. Transgenic mice with conditional deletion of Cx43 in smooth muscle cells (SMC) were generated. Histological investigations by immunofluorescence analyses and organ-bath recordings to assess the contractility of intestinal tissue strips were carried out. Measurements of gastrointestinal transit and of the visceromotor response by utilizing a standardized colorectal distension model to quantify alterations of visceral sensory function were also performed in SMC-specific Cx43 null mice and control littermates. Histologically, we found thickening of the tunica muscularis and a 13-fold increase of neutrophil infiltration of the gastrointestinal wall of SMC-specific Cx43 null mice. These animals also exhibited a decrease of 29% in gastrointestinal transit time. In contrast, the visceromotor response to a standardized colorectal distension was elevated, as was the contractility in SMC-specific Cx43 null mice, compared with controls. Thus, SMC-specific ablation of Cx43 in mice leads to morphological and functional alterations of the intestinal tunica muscularis, to gastrointestinal motor dysfunction and to altered visceral sensory function. This study was supported by a grant from the German Research Association (Wi 270/25-1,2) to K.W. and in part by the IFORES program of the University Hospital, Essen, Germany.     

Value of apoptin’s 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells

Apoptin, a protein of the chicken anemia virus (CAV), consists of 121 amino acids (aa) and represents a novel, potentially tumor-specific therapeutic and diagnostic agent. The C-terminal part of Apoptin (aa 81–121) is believed to contain a bipartite nuclear localization signal (NLS) (NLS1: aa 82–88 and NLS2: aa 111–121), which is only active in tumor cells after phosphorylation of threonine¹⁰⁸ by tumor-specific cytoplasmic phosphokinases. Furthermore, a nuclear export signal (NES) (aa 97–105) seems to enable nuclear export of Apoptin only in healthy cells. The specificity for tumor cell nuclei also applies to the truncated C-terminal part of Apoptin (aa 81–121), which therefore represents a highly attractive peptide sequence for peptide synthesis. Here we describe for the first time the synthesis of fluorescein isothiocyanate (FITC)- and Dansyl-labelled conjugates containing this C-terminal part of Apoptin, with either phosphorylated or nonphosphorylated threonine¹⁰⁸. The phosphorylated conjugates were synthesized in an attempt to achieve nuclear accumulation in healthy cells, which lack cytoplasmic tumor-specific phosphokinases. Surprisingly, all the conjugates accumulated rapidly within the cell nuclei of both tumor and non-tumor cells from the bladder, brain and prostate and led to cell death. By coupling Apoptin^81–121 to FITC and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at either the C- or N-terminus we could exlude that the coupling site is decisive for tumor cell-specific nuclear localization. The labels FITC, DOTA and Dansyl were not responsible for cell death in healthy cells because cell death was not prevented by using an unlabelled Apoptin^81–121 peptide. Cellular and nuclear uptake of the FITC-labelled Apoptin^81–121 peptide was almost completely abolished after altering the NLS2 (replacement of five arginines with serines).     

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.     

Genetic Evidence That Chain Length and Branch Point Distributions Are Linked Determinants of Starch Granule Formation in Arabidopsis

The major component of starch is the branched glucan amylopectin. Structural features of amylopectin, such as the branching pattern and the chain length distribution, are thought to be key factors that enable it to form semicrystalline starch granules. We varied both structural parameters by creating Arabidopsis (Arabidopsis thaliana) mutants lacking combinations of starch synthases (SSs) SS1, SS2, and SS3 (to vary chain lengths) and the debranching enzyme ISOAMYLASE1-ISOAMYLASE2 (ISA; to alter branching pattern). The isa mutant accumulates primarily phytoglycogen in leaf mesophyll cells, with only small amounts of starch in other cell types (epidermis and bundle sheath cells). This balance can be significantly shifted by mutating different SSs. Mutation of SS1 promoted starch synthesis, restoring granules in mesophyll cell plastids. Mutation of SS2 decreased starch synthesis, abolishing granules in epidermal and bundle sheath cells. Thus, the types of SSs present affect the crystallinity and thus the solubility of the glucans made, compensating for or compounding the effects of an aberrant branching pattern. Interestingly, ss2 mutant plants contained small amounts of phytoglycogen in addition to aberrant starch. Likewise, ss2ss3 plants contained phytoglycogen, but were almost devoid of glucan despite retaining other SS isoforms. Surprisingly, glucan production was restored in the ss2ss3isa triple mutants, indicating that SS activity in ss2ss3 per se is not limiting but that the isoamylase suppresses glucan accumulation. We conclude that loss of only SSs can cause phytoglycogen production. This is readily degraded by isoamylase and other enzymes so it does not accumulate and was previously unnoticed.Starch, the major storage carbohydrate in plants, is composed of two α-1,4- and α-1,6-linked glucan polymers: moderately branched amylopectin and predominantly linear amylose. Amylopectin, which constitutes approximately 80% of most starches, is synthesized by three enzyme activities. Starch synthases (SSs) transfer the glucosyl moiety of ADP-Glc to a glucan chain, forming a new α-1,4 glucosidic linkage, extending the linear chains. Branching enzymes (BEs) cleave some α-1,4 linkages and reattach chains of six Glc units or more via α-1,6 linkages, creating branch points. Debranching enzymes (DBEs) hydrolyze some of these branches, tailoring the structure of the polymer. However, the way in which the individual enzymes work together to create crystallization-competent amylopectin remains unclear.The coordinated actions of SSs, BEs, and DBEs are thought to produce a glucan with a tree-like architecture in which the branch points are nonrandomly positioned. According to models of amylopectin, clusters of unbranched chain segments are formed. Within these clusters, adjacent chains form double helices, which align in parallel giving rise to crystalline lamellae. These alternate with amorphous lamellae containing the branch points and chain segments that span the clusters (Zeeman et al., 2010). In the context of this amylopectin model, glucan chains can be categorized according to their length and connection to other chains. The A chains are external chains that do not carry other branches. The B chains carry one or more branches (either an A chain or another B chain) and have both external and internal segments. The B chains can span one or more clusters (e.g. a B₁ chain spans one cluster). The C chain is the single chain that has a reducing end (Manners, 1989). The A chains tend to be the shortest, having an average chain length (ACL) of 12 to 16, depending on the species (Hizukuri, 1986). Together with the B₁ chains, the A chains are thought to make up the crystalline clusters. Longer chains such as B₂ chains (ACL 20–24) or B₃ chains (ACL 42–48) are presumed to connect clusters (Hizukuri, 1986). Amylose is a distinct polymer synthesized within the amylopectin matrix by granule-bound SS (Tatge et al., 1999). Mutants lacking granule-bound SS also lack amylose but still make starch granules, showing that amylose synthesis is not required for this (Zeeman et al., 2010).The structural properties of amylopectin contrast with those of glycogen, the Glc polymer synthesized in organisms such as fungi, animals, and most bacteria. Glycogen also consists of α-1,4-linked Glc chains with α-1,6-linked branches, but differs in three major ways from amylopectin. First, its external branches are considerably shorter (6–8 Glc units compared with 12–16 in amylopectin). Second, the branch frequency (10%) is twice as high as in amylopectin. Third, its branch points are assumed to be distributed homogeneously, whereas branching in amylopectin is thought to be nonhomogeneous. These differences prevent the formation and parallel alignment of double helices in glycogen, rendering it soluble. Glycogen synthesis requires only a single glycogen synthase enzyme and a single glycogen BE, whereas several SS and BE isoforms are involved in amylopectin synthesis. In Arabidopsis (Arabidopsis thaliana), there are four SSs (SS1–SS4) and two BEs (BE2 and BE3; Li et al., 2003; Streb and Zeeman, 2012). In addition, Arabidopsis has three DBEs. ISOAMYLASE1-ISOAMYLASE2 (hereafter referred to simply as ISA), a heteromultimeric enzyme composed of the two subunits ISA1 and ISA2, is implicated in amylopectin synthesis (Delatte et al., 2005). The other two DBEs, ISA3 and LIMIT DEXTRINASE (LDA), are implicated in starch degradation (Delatte et al., 2006).Loss of specific SS isoforms has different effects on the starch amount, amylopectin chain length distribution (CLD), and starch granule morphology, suggesting distinct functions for each isoform. For example, amylopectin from SS1-deficient mutants of Arabidopsis (Delvallé et al., 2005; Szydlowski et al., 2011) and rice (Oryza sativa; Fujita et al., 2006) has fewer chains with a degree of polymerization (DP; i.e. chain length) between 8 and 12 and more chains with a DP between 17 and 20 compared with the wild-type starches. This is consistent with in vitro data for the maize (Zea mays; Commuri and Keeling, 2001) and rice SSI enzymes (Fujita et al., 2006), which preferentially elongate short chains of DP 6 or 7 up to a length of DP 10. This indicates that SSI functions to elongate the short chains created by BEs by a few Glc units (Commuri and Keeling, 2001; Delvallé et al., 2005). Comparable studies in SS2-deficient mutants reveal amylopectin with more chains with DP 6 to 11, but depletion in chains with DP 13 to 20 compared with the corresponding wild-type amylopectins. Thus, SS2 is suggested to elongate shorter chains (e.g. those made by SS1) to a length of between DP 13 and 20 (Edwards et al., 1999; Yamamori et al., 2000; Umemoto et al., 2002; Morell et al., 2003; Zhang et al., 2004, 2008). SS3 was proposed to be important for the generation of long, cluster-spanning chains (Jeon et al., 2010; Tetlow and Emes, 2011), as well as contributing to A chain and B₁ chain elongation (Edwards et al., 1999; Zhang et al., 2005, 2008). By contrast, SS4 appears to have a specialized role in initiating or coordinating granule formation (Roldán et al., 2007; Crumpton-Taylor et al., 2012, 2013). Arabidopsis ss4 mutants have just one round starch granule per chloroplast rather than five or more lenticular granules observed in the wild type.Partial loss of BE activity in maize (Stinard et al., 1993), rice (Mizuno et al., 1993), and potato (Solanum tuberosum; Schwall et al., 2000) leads to starches with high apparent amylose, most likely caused by the accumulation of less frequently branched amylopectin. A total lack of branching activity in Arabidopsis be2be3 mutants, however, abolishes starch production. Instead, maltose accumulates, suggesting that linear glucans are produced, but degraded by α- and β-amylases (Dumez et al., 2006).Loss of DBE of the ISA1 class causes a dramatic phenotype, with production of a soluble glucan (phytoglycogen) in place of starch. This has been observed in starch-synthesizing tissues of several species, including Chlamydomonas reinhardtii cells (Mouille et al., 1996), Arabidopsis leaves (Delatte et al., 2005; Wattebled et al., 2005), and the endosperms of maize (Zea Mays; James et al., 1995), rice (Oryza sativa; Nakamura et al., 1997), and barley (Hordeum vulgare) seeds (Burton et al., 2002). Phytoglycogen has structural similarities to glycogen in that both are water soluble and have a higher branch frequency than amylopectin. Accordingly, it was proposed that the trimming of glucans produced by SS and BE isoforms by ISA1 removes branches that interfere with the formation of secondary and tertiary structures (i.e. organized arrays of double helices), thereby facilitating amylopectin biosynthesis and crystallization (Ball et al., 1996). Compared with ISA1, the other two DBEs (LDA and ISA3) have different substrate specificities, both preferring substrates with short outer chains, such as β-limit dextrins, suggesting that their role is primarily in starch degradation. Consistently, mutating these genes in Arabidopsis causes a starch-excess phenotype rather than phytoglycogen accumulation (Delatte et al., 2006).Although it is now widely accepted that a degree of debranching occurs to control branch number and positioning in amylopectin, the importance of this for crystalline starch production is still uncertain. Several studies have shown that some cell types in isa1-deficient mutants still produce some starch (e.g. epidermal and bundle sheath cells in Arabidopsis mutants; Delatte et al., 2005), indicating that other factors can also affect the partitioning between phytoglycogen and starch.No starch granules are made in the Arabidopsis isa1isa2isa3lda quadruple mutant, which lacks all three DBEs (Streb et al., 2008). Although suggestive of redundancy between the DBEs, the loss of each enzyme has distinct effects on amylopectin or phytoglycogen structure, consistent with their different substrate specificities. Furthermore, the loss of starch granules in isa1isa2isa3lda was shown to be at least partly due to the actions of α-amylase; typical α-amylolytic products (short malto-oligosaccharides) accumulated alongside phytoglycogen. Mutation of the gene encoding the chloroplastic α-AMYLASE3 (AMY3) eliminated these short malto-oligosaccharides and restored starch granule biosynthesis in all cell types examined. This unexpected result showed that crystalline glucans can be produced in the absence of DBE activity, despite an altered branching pattern. Streb et al. (2008) proposed that AMY3 shortens external chains of the glucans made by SSs and BEs so that they cannot form double helices with their neighbors. This idea is consistent with models for amylopectin, in which a suitable CLD is a critical factor in the formation of the secondary and higher-order crystalline structures (Gidley and Bulpin, 1987; Pfannemüller, 1987). Thus, factors that affect the CLD, such as a failure to sufficiently elongate new branches or concomitant chain degradation by amylases, should also affect crystallinity. Indeed, early studies of maize mutants (that were subsequently shown to be affected in DBE and SS activities) reported that loss of SS in a DBE mutant background altered the ratio of starch to phytoglycogen compared with the DBE mutants alone (Cameron and Cole, 1954; Creech, 1965).The aim of this work was to use genetics to systematically vary both branch point position and chain lengths and determine the impact on glucan amount, structure, and starch granule formation in Arabidopsis. We analyzed mutants lacking combinations of SSs (to vary chain lengths) in the absence of the debranching enzyme ISA1-ISA2 (to change branch point distribution/frequency). This revealed that the length of external chains is a key factor in the production of a crystallization-competent glucan. Remarkably, our results also provide evidence for phytoglycogen production due to mutations just in SSs. Our results indicate that this phenomenon is largely masked by the presence of ISA1-ISA2, which degrades the aberrant glucan instead of trimming it to amylopectin.     

Fatty acylation promotes fusion of transport vesicles with Golgi cisternae

Two different methods, stimulation of transport by fatty acyl-coenzyme A (CoA) and inhibition of transport by a nonhydrolyzable analogue of palmitoyl-CoA, reveal that fatty acylation is required to promote fusion of transport vesicles with Golgi cisternae. Specifically, fatty acyl-CoA is needed after the attachment of coated vesicles and subsequent uncoating of the vesicles, and after the binding of the NEM-sensitive fusion protein (NSF) to the membranes, but before the actual fusion event. We therefore suggest that an acylated transport component participates, directly or indirectly, in membrane fusion.