Silencing of HIV by hairpin-loop-structured DNA oligonucleotide

We describe inhibition of HIV replication by a partially double-stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple-helix formation. Upon treatment of HIV-infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN-mediated effect was highly sequence-specific. The ODN against HIV-IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV-BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.     

Aberrant gata-3 expression in human pancreatic cancer.

Gata-3 has been shown to specifically alter its expression patterns in different types of cancers. Recent evidence suggests that an interference of Gata-3 exists in the TGF-beta signaling pathway. To determine the role of Gata-3 in pancreatic cancer, pancreatic cancer samples were analyzed in comparison to normal pancreatic tissues. Furthermore, four different pancreatic cancer cell lines with different alterations of the TGF-beta pathway were studied. To evaluate if a potential relationship with TGF-beta signaling pathway exists, we correlated mRNA expression levels with the expression of TGF-betas, TGF-beta receptors, and Smad-3. Finally, we analyzed the influence of TGF-beta on Gata-3 expression in vitro. All pancreatic cancer samples demonstrated a marked overexpression of Gata-3 mRNA and protein. Immunohistochemical staining revealed strong and persistent cytoplasmic Gata-3 immunoreactivity in cancer cells. In an electrophoretic mobility shift assay, a disturbed nuclear translocation was confirmed. The expression of Gata-3 showed a significant correlation with the expression of TGF-betas, TGF-beta receptors, and Smad-3. TGF-beta responsive cell lines showed a downregulation of Gata-3 mRNA upon TGF-beta exposure, whereas in TGF-beta-unresponsive cell lines, Gata-3 mRNA expression persisted at high levels. Furthermore, strong specific upregulation of Gata-3 impaired nuclear translocation and its cooperative action with the TGF-beta pathway, suggesting that Gata-3 plays a central role in human pancreatic cancer.     

Culture-Independent Characterization of the Digestive-Tract Microbiota of the Medicinal Leech Reveals a Tripartite Symbiosis

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the α-, γ-, and δ-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.     

The Two Plastidial Starch-Related Dikinases Sequentially Phosphorylate Glucosyl Residues at the Surface of Both the A- and B-Type Allomorphs of Crystallized Maltodextrins But the Mode of Action Differs

In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of monophosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.In terms of quantity, starch is one of the most prominent photosynthesis-derived products. The global starch production by land plants has been estimated to be approximately 2,850 million tons per year (Burrell, 2003). Starch is highly relevant for nutrition in animals and humans, but it is also used for many industrial applications, such as additives in paper or textiles and in pharmacy products as well. In addition, starch appears to be increasingly important as a photosynthesis-based renewable energy source that can be converted into technologically relevant products such as bioethanol and hydrogen (Hannah and James, 2008; Zhang et al., 2008).Native starch is formed as a water-insoluble particle called a granule that is thought to comprise two types of polyglucans, amylopectin and amylose. The latter is an almost unbranched α-1,4-glucan and usually is the minor constituent of the starch particle, accounting for 10% to 35% of the total starch dry weight (Ball, 2000). However, in some mutants, the relative amylose content is strongly diminished, resulting in an essentially amylose-free starch (such as in the waxy mutant of maize [Zea mays]), or, alternatively, it is increased, forming up to 70% of the starch mass (e.g. in the amylose extender mutant from maize; Gérard et al., 2001). Nevertheless, in wild-type starches, amylopectin typically is the major constituent that also is essential for the molecular organization of the glucans within the entire starch granule (Ball and Morell, 2003). Like glycogen, amylopectin is a branched α-glucan with 4% to 6% of the inter-Glc linkages being α-1,6-bonds (Ball, 2000); however, as opposed to glycogen, the branching points occur as intramolecular clusters. Due to the length distribution of the side chains and the clustering of the branching points, neighboring glucan chains are capable of forming highly ordered double helices (Smith, 2001; Zeeman et al., 2002).As revealed by x-ray diffraction analysis, two major native starch structures are known that differ in the arrangement of the double helices. The A-type allomorph, which is typical of wild-type cereal starches but also occurs in lower plants, is more compact, as compared with the B type, and consists of flat layers of double helices. By contrast, in the B-type allomorph, six double helices are thought to surround a central cavity that is filled with water molecules. The B-type allomorph is found in starch synthesized by dicotyledonal storage organs, such as potato (Solanum tuberosum) tubers, in some high-amylose starches from cereal mutants (Gallant et al., 1997; Gérard et al., 2001), and in assimilatory starches from potato and Arabidopsis (Arabidopsis thaliana) as well (Hejazi et al., 2008). Legume starches are believed to represent another allomorph that is designated the C type. However, this allomorph is actually a mixture of both the A- and B-type crystallites within a single native starch particle rather than a third distinct type of the double helical arrangement (Imberty et al., 1991; Bogracheva et al., 2001).It should be noted that both the A- and B-type allomorphs of native starch granules often contain, as a minor constituent, an additional crystal structure designated the V type. Unlike the A- and B-type allomorphs, the V type is assumed to arise from single amylose helices, some of which are complexed with endogenous granular lipids. When estimated for the dry state, the V-type crystal structure accounts for only a small percentage of the total starch granule crystallinity (Lopez-Rubio et al., 2008).The physical structure of the native starch particle is likely to have important biochemical implications, as it affects the performance of carbohydrate-active enzymes and, thereby, the transition of carbohydrates from the solid phase to the soluble phase. This conclusion has been reached by in vitro experiments demonstrating that the pancreas α-amylase hydrolyzes A-type starch faster than the B-type counterpart (Gérard et al., 2001).Another metabolically important feature of amylopectin is the occurrence of covalent modification by phosphate esters that are found in a small proportion of the glucosyl residues. Most frequently phosphorylation occurs at the C6 position of the glucosyl residue, but C3 and, to a minor extent, C2 can also be esterified (Hizukuri et al., 1970). Recently, evidence has been presented that the esterification of the C6 and C3 positions of glucosyl residues differs in the structural effects on the neighboring inter-Glc bonds (Hansen et al., 2009). Phosphorylation at C6 is mediated by the recently identified α-glucan, water dikinase (GWD; EC 2.7.9.4), which utilizes ATP as dual phosphate donor and three distinct acceptors, two of which are sequentially used. The enzyme transfers the terminal phosphate group to water (thereby forming orthophosphate) and the β-phosphate group first to a conserved His residue within the catalytic domain of the monomeric GWD and, subsequently, to the C6 target of the glucosyl residue to be phosphorylated (Ritte et al., 2002, 2006). Phosphorylation at C3 is catalyzed by a second dikinase, designated phosphoglucan, water dikinase (PWD; EC 2.7.9.5; Ritte et al., 2006). The amino acid sequence of the catalytic (C-terminal) domain of PWD shares similarity with that of GWD, and in principle, the PWD-mediated catalysis follows the same mode of action as GWD, including the transient autophosphorylation at a conserved His residue (Baunsgaard et al., 2005; Kötting et al., 2005). However, PWD deviates from GWD in the amino acid sequence of the N-terminal domain, especially in the carbohydrate-binding region. PWD possesses a single carbohydrate-binding module that has been grouped into the family CBM20 (Machovič and Janaček, 2006a, 2006b). By contrast, the N-terminal domain of GWD contains two putative carbohydrate-binding motifs similar to those of an α-amylase that presumably is located in the chloroplasts (Yu et al., 2005). However, the structure of these motifs is still not known; therefore, a sequence-based prediction of the actual carbohydrate target is not yet possible.GWD- and PWD-deficient Arabidopsis mutants possess to some extent similar but not equal phenotypes. Leaves of GWD-deficient lines (which contain essentially unchanged levels of functional PWD) have starch levels that are at least five times higher than those of the wild type and remain high even after prolonged darkness. Growth of the entire plant is strongly compromised. The phenotype of PWD-deficient mutants (which express functional GWD) is less severe, as growth is only slightly diminished and transitory starch levels are elevated but not as strongly as in the GWD-deficient lines. Mutants lacking functional PWD can degrade transitory starch, but net degradation occurs at a lower rate as compared with wild-type plants (Kötting et al., 2005). These data clearly indicate that, in vivo, PWD cannot substitute for GWD and that glucosyl 6-phosphate residues are involved in a more strict control of the starch turnover as compared with the C3 phosphate esters.When considering the metabolic function(s) of starch phosphorylation, it should be noted that phosphorylation occurs during both net starch synthesis and degradation, although the rates of phosphorylation are likely to be different (Nielsen et al., 1994; Ritte et al., 2004). It is reasonable, therefore, to assume that starch phosphorylation exerts an important role in the entire transitory starch metabolism, rather than being functional only during the degrading process (and, consequently, the starch-related dikinases cannot, in a strict sense, be considered as “starch-degrading enzymes”).Depending on the botanical source, the degree of starch phosphorylation varies strongly. In potato tuber starch, approximately 0.1% to 0.5% of the glucosyl residues are phosphorylated (Ritte et al., 2002), and this value is considered to be indicative of a high level of phosphorylation. By contrast, cereal starches contain a far lower relative phosphate content that often is close to the limit of detection (approximately 0.002%; Glaring et al., 2006). In principle, these differences could be due to different rates of phosphorylation, as catalyzed by the two starch-related dikinases, and this assumption seems to be supported by the observation that, in general, starches of the B-type allomorph appear to have a higher degree of phosphorylation as compared with those of the A-type allomorph. If so, the dikinases may preferentially act on the B-type allomorph. Alternatively, the phosphorylation catalyzed by the two dikinases could be balanced by counteracting phosphatases, such as SEX4. This plastidial enzyme has been shown to act as a (phospho)glucan phosphatase that is involved in leaf starch metabolism (Kötting et al., 2009). If antagonistic enzyme activities are taken into consideration, the actual level of starch phosphorylation is determined by the rate of both phosphorylation and the subsequent hydrolysis of phosphate esters and, consequently, does not necessarily reflect the action of the starch-related dikinases.Recently, crystallized maltodextrins (MD_cryst) have been prepared that, by using x-ray diffraction, were identified as being the B-type allomorph and to possess a highly ordered structure (which exceeds that of native starch granules). MD_cryst have been applied as a substrate for a recombinant GWD from potato. Using a carefully optimized assay, the rate of phosphorylation was by far higher than that observed with any other carbohydrate substrate, such as native starch granules or starch-derived polysaccharides. By contrast, solubilization by heat treatment of the MD_cryst almost completely abolished the activity of GWD. Phosphorylation resulted in the formation of singly, doubly, and triply phosphorylated glucans and favored the solubilization of both neutral glucans and phosphoglucans (Hejazi et al., 2008). Recombinant PWD also phosphorylated MD_cryst, provided the MD_cryst had been prephosphorylated by GWD and were not solubilized by heat treatment (Hejazi et al., 2008).Because of the high phosphorylation rates and the phosphorylation pattern obtained, MD_cryst are a suitable model carbohydrate that mimics phosphorylation-relevant features of highly ordered regions within the native starch granule. It allows study of the action of the two starch-related dikinases and the transition of carbohydrates from the solid to the soluble state without any other starch-related enzyme being required.Until now, only the B-type allomorph of the MD_cryst has been applied as substrate of the two dikinases. Using native starch granules as a target, the rates of phosphorylation as obtained with recombinant GWD varied largely within the B-type allomorph (Hejazi et al., 2008); therefore, it is reasonable to assume that additional but largely unknown features of the native starch granule also strongly affect the action of GWD. This implies that any preference or specificity of the starch-related dikinases for a given allomorph can be analyzed most convincingly if MD_cryst preparations representing both the B- and A-type allomorphs are available.In this study, we used two MD_cryst preparations that are indistinguishable in their oligoglucan patterns but differ in the physical arrangement of the double helices and represent the highly ordered A- and B-type allomorphs. Using these two MD_cryst preparations, we analyzed the action of the two starch-related dikinases. The size distribution of the MD_cryst particles has been determined using the Coulter counter, and surface properties of both allomorphs were monitored by scanning electron microscopy. Thermal stability of the two allomorphs was analyzed by measuring the temperature dependence of light scattering. Finally, the phosphorylation-dependent solubilization of both allomorphs and the transition of (phospho)glucans into the soluble state have been studied.     

Symbiont Succession during Embryonic Development of the European Medicinal Leech,Hirudo verbana

The European medicinal leech, Hirudo verbana, harbors simple microbial communities in the digestive tract and bladder. The colonization history, infection frequency, and growth dynamics of symbionts through host embryogenesis are described using diagnostic PCR and quantitative PCR. Symbiont species displayed diversity in temporal establishment and proliferation through leech development.The hermaphroditic European medicinal leech (Hirudo spp.) is one of the most extensively examined animal models in neurophysiological, developmental, and behavioral studies (14) and has recently been used as a naturally occurring simple model for beneficial symbioses (5, 13). A fundamental question of microbial symbioses is how to determine the transmission mode of the symbionts between generations. Hirudo verbana reproduces by depositing eggs, which are surrounded by a cocoon. The cocoon is secreted from glandular cells of the parental mouth and usually contains 5 to 25 eggs. Each individual egg is encased by a self-enclosed vitelline membrane, referred to as the larval sac, and is bathed in a nutritious albumenous fluid (14). Complete embryonic development occurs within the cocoon and is composed of two distinct life stages, cryptolarva and juvenile. The cryptolarva transitions into a juvenile approximately midway into embryogenesis. The temporal acquisition of morphological attributes during embryonic development have been well described (3, 12, 16) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Paradigm of percent embryonic development (% ED) of the European medicinal leech, H. verbana, relative to the acquisition of digestive tract features. At 20 ± 1°C, 24 h is equivalent to 3.33% ED, with complete embryogenesis (spanning from cocoon deposition to the emergence of adult-like juveniles) requiring approximately 30 h. Staging scheme based on references 3 and 12. *, sampling time point; PD, days postcocoon deposition; prs, pairs; d, days. (Adapted from reference 12 with permission of John Wiley & Sons. Copyright 1998 Wiley-Liss, Inc.)The medicinal leech houses distinct microbial communities within its digestive tract and secretory bladders. Culturing and culture-independent profiling of the European medicinal leech, H. verbana, through fluorescence in situ hybridization, study of 16S rRNA gene clone libraries, and terminal restric-tion length polymorphism, revealed a simple and stable microbial community within the adult midgut (2, 4, 7, 8, 18). The gammaproteobacterium Aeromonas veronii and a member of the Bacteroidetes, Rikenella, were identified as consistent and dominant extracellular residents of the medicinal leech crop and intestinum. An early culture-based study detected a bacterium that is now considered to be A. veronii in the cocoon albumen and in young leeches after hatching (1). In previous electron microscopy work investigating the embryonic development of the bladders, intracellular bacteria were detected within the bladder wall and extracellular bacteria within the lumen (2, 16, 17). A recent study revealed that this microbiota is organized in distinct layers and is composed of the deltaproteobacterium Bdellovibrio, betaproteobacteria Comamonas and Sterolibacterium, members of the Bacteroidetes, Sphingobacterium and Niabella, and alphaproteobacterium Ochrobactrum spp. (10). Although the microbial constituents of the adult H. verbana digestive tract have been previously characterized, the succession, inoculum sizes, frequency of infection, and growth dynamics of these symbiont species during embryogenesis remain to be described.Putative functional roles for the crop/intestinum symbionts of the leech host include aiding in digestion, provisioning essential nutrients to the host, which are lacking in the blood meal (14), and preventing the establishment of foreign microbiota (1, 15). The symbionts localized in the bladders are suspected to play a role in the recycling of host metabolic waste into ammonia (10). The digestive tract symbionts may also display nutritional syntrophy, and possibly, A. veronii primes the host''s digestive tract to enable the establishment and persistence of the obligate anaerobic Rikenella-like bacterium, thereby contributing to the selection of the future microbiota (reviewed in reference 13). This paper details the microbial colonization patterns relative to H. verbana embryogenesis using a combination of species-specific diagnostic PCR and quantitative PCR (qPCR) analyses.     

Leukocyte Redistribution: Effects of Beta Blockers in Patients with Chronic Heart Failure

Background

Overproduction of pro-inflammatory cytokines is a well established factor in the progression of chronic heart failure (CHF). Changes in cellular immunity have not been widely studied, and the impact of standard medication is uncertain. Here we investigate whether a leukocyte redistribution occurs in CHF and whether this effect is influenced by beta-blocker therapy.

Methodology

We prospectively studied 75 patients with systolic CHF (age: 68±11 years, left ventricular ejection fraction 32±11%, New York Heart Association class 2.5±0.7) and 20 age-matched healthy control subjects (age: 63±10 years). We measured the response of cells to endotoxin exposure in vitro, analysed subsets of lymphocytes using flow cytometry, and assessed plasma levels of the pro-inflammatory markers interleukin 1, 6, tumor necrosis factor-α, and soluble tumor necrosis factor receptors 1 and 2.

Principal findings

While no differences in the number of leukocytes were noted between patients with CHF and healthy controls, we detected relative lymphopenia in patients with CHF (p<0.001 vs. control), mostly driven by reductions in T helper cells and B cells (both p<0.05). The number of neutrophils was increased (p<0.01). These effects were pronounced in patients who were beta-blocker naïve (32% of all patients with CHF). Increased plasma levels of soluble tumor necrosis receptor-1 correlated with the relative number of lymphocyte subsets.

Conclusions

In patients with CHF, we detected a redistribution of leukocyte subsets, i.e. an increase in neutrophils with relative lymphopenia. These effects were pronounced in patients who were beta-blocker naïve. The underlying mechanism remains to be elucidated.     

The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans

The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of α-glucans from the insoluble to the soluble state.The metabolism of starch, the most prominent storage carbohydrate in plants, is assumed to require approximately 30 to 40 distinct (iso)enzymes (Deschamps et al., 2008), but, presumably, the list of the starch-related proteins is not yet complete. Several novel proteins (and protein functions) essential for the normal starch metabolism have recently been identified among which are two α-glucan phosphorylating dikinases. One dikinase (glucan, water dikinase [GWD], EC 2.7.9.4) utilizes ATP as dual phosphate donor and esterifies the C6 position of amylopectin-related glucosyl residues, whereas the other dikinase (phosphoglucan, water dikinase [PWD], EC 2.7.9.5) selectively transfers the β-phosphate group from ATP to the C3 position of glucosyl residues (Ritte et al., 2006).Two other previously unknown starch-related enzymes were designated as SEX4 protein (EC 3.1.3.48; At3g52180; previous designations PTPKIS1 and DSP4) and as Like Sex Four1 (LSF1) protein (At3g01510; previously named PTPKIS2; Comparot-Moss et al., 2010). Both proteins are predicted to contain a noncatalytic carbohydrate-binding module (CBM; Boraston et al., 2004; Shoseyov et al., 2006) and a catalytic dual-specificity phosphatase (DSP) domain. The latter is shared by the large family of DSPs that dephosphorylate distinct target phosphoproteins both at phosphotyrosine and phosphoserine/phosphothreonine residues. Some DSPs also act on various nonproteinaceous substrates, such as phospholipids or phosphorylated polyglycans (Pulido and Hooft van Huijsduijnen, 2008).Arabidopsis (Arabidopsis thaliana) mutants lacking a functional SEX4 protein contain both elevated starch levels and significant amounts of soluble phosphooligoglucans that are below the limit of detection in wild-type plants and probably originate from starch. However, the precise biochemical function of SEX4 is far from being clear (Kötting et al., 2009). The phenotype of the SEX4-deficient mutant is complex: Transitory starch possesses an elevated amylose-to-amylopectin ratio but the phosphate content of amylopectin is not increased. It has been hypothesized that SEX4 and LSF1 selectively hydrolyze C6- and C3-phosphate esters, respectively, but experimental evidence is lacking (Kötting et al., 2009). Likewise, it is unknown whether SEX4 preferentially acts on particulate starch or on soluble phosphoglucans.Crystalline maltodextrins (MD_cryst) have recently been introduced as a model mimicking some structural features of the native starch granules (Hejazi et al., 2008). They can be crystallized as either the A- or the B-type allomorph (Gallant et al., 1997; Gérard et al., 2001). Recombinant StGWD phosphorylates both maltodextrin allomorphs with a far higher rate than native starch granules and thereby initiates solubilization of both phosphorylated and nonphosphorylated maltodextrins. In vitro both allomorphs act also as substrate for PWD provided a prephosphorylation by GWD (Hejazi et al., 2009).In this study, we used the prephosphorylated A- and B-type allomorphs of MD_cryst to study biochemical functions of AtSEX4. As highly ordered α-glucans are the preferred sites of the dikinase-mediated phosphorylation, we designed experiments to answer the following questions: Does SEX4 preferentially act on phosphorylated insoluble or soluble glucans? If insoluble α-glucans are the preferred substrate, does the phosphatase distinguish between the A- and the B-type allomorph? Does SEX4 preferentially or selectively hydrolyze C6-phosphate esters? Does SEX4 also interact with nonphosphorylated oligoglucans? Finally, assuming that SEX4 acts on insoluble phosphoglucans, does the removal of phosphate esters affect the phase transition and/or the physical order of the glucans?