Maize opaque5 encodes monogalactosyldiacylglycerol synthase and specifically affects galactolipids necessary for amyloplast and chloroplast function.

The maize (Zea mays) opaque5 (o5) locus was shown to encode the monogalactosyldiacylglycerol synthase MGD1. Null and point mutations of o5 that affect the vitreous nature of mature endosperm engendered an allelic series of lines with stepwise reductions in gene function. C(18:3)/C(18:2) galactolipid abundance in seedling leaves was reduced proportionally, without significant effects on total galactolipid content. This alteration in polar lipid composition disrupted the organization of thylakoid membranes into granal stacks. Total galactolipid abundance in endosperm was strongly reduced in o5(-) mutants, causing developmental defects and changes in starch production such that the normal simple granules were replaced with compound granules separated by amyloplast membrane. Complete loss of MGD1 function in a null mutant caused kernel lethality owing to failure in both endosperm and embryo development. The data demonstrate that low-abundance galactolipids with five double bonds serve functions in plastid membranes that are not replaced by the predominant species with six double bonds. Furthermore, the data identify a function of amyloplast membranes in the development of starch granules. Finally, the specific changes in lipid composition suggest that MGD1 can distinguish the constituency of acyl groups on its diacylglycerol substrate based upon the degree of desaturation.

Intra-prostatic tumour evolution, steps in metastatic spread and histogenomic associations revealed by integration of multi-region whole-genome sequencing with histopathological features.

BACKGROUND: Extension of prostate cancer beyond the primary site by local invasion or nodal metastasis is associated with poor prognosis. Despite significant research on tumour evolution in prostate cancer metastasis, the emergence and evolution of cancer clones at this early stage of expansion and spread are poorly understood. We aimed to delineate the routes of evolution and cancer spread within the prostate and to seminal vesicles and lymph nodes, linking these to histological features that are used in diagnostic risk stratification. METHODS: We performed whole-genome sequencing on 42 prostate cancer samples from the prostate, seminal vesicles and lymph nodes of five treatment-naive patients with locally advanced disease. We spatially mapped the clonal composition of cancer across the prostate and the routes of spread of cancer cells within the prostate and to seminal vesicles and lymph nodes in each individual by analysing a total of > 19,000 copy number corrected single nucleotide variants. RESULTS: In each patient, we identified sample locations corresponding to the earliest part of the malignancy. In patient 10, we mapped the spread of cancer from the apex of the prostate to the seminal vesicles and identified specific genomic changes associated with the transformation of adenocarcinoma to amphicrine morphology during this spread. Furthermore, we show that the lymph node metastases in this patient arose from specific cancer clones found at the base of the prostate and the seminal vesicles. In patient 15, we observed increased mutational burden, altered mutational signatures and histological changes associated with whole genome duplication. In all patients in whom histological heterogeneity was observed (4/5), we found that the distinct morphologies were located on separate branches of their respective evolutionary trees. CONCLUSIONS: Our results link histological transformation with specific genomic alterations and phylogenetic branching. These findings have implications for diagnosis and risk stratification, in addition to providing a rationale for further studies to characterise the genetic changes causally linked to morphological transformation. Our study demonstrates the value of integrating multi-region sequencing with histopathological data to understand tumour evolution and identify mechanisms of prostate cancer spread.

Functional interactions between starch synthase III and isoamylase-type starch-debranching enzyme in maize endosperm.

This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1(-) mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.

Mindful Movement Intervention Applied to at Risk Urban School Children for Improving Motor, Cognitive, and Emotional-Behavioral Regulation.

Objectives: Preliminary evidence has supported the notion that mindful movement-based practices may offer benefits for self-regulation, particularly for vulnerable children. However, this evidence has principally stemmed from subjective assessments of behavioral change, leaving the underlying mechanisms undetermined. The present study aimed to investigate the efficacy of an in-school mindful movement intervention (MMI) for at-risk children within an urban public school for enhancing motor, cognitive, and emotional-behavioral regulation, including control of disruptive and inattentive behaviors characteristic of ADHD. Method: Participants included 38 (age 7-8 years) children who received twice weekly, in-school MMI, including a modified Tai Chi sequence, yoga and biomechanical warm-ups, imaginative play, and reflection. Parent and teacher ratings of disruptive behaviors, and objective measures of motor and cognitive control, were collected at baseline and after 5 months of MMI. Results: Significant improvements in teacher ratings of inattentive, hyperactive/impulsive, oppositional, and other disruptive behaviors were observed. Significant improvements were also observed for objective measures of both cognitive control and motor control with particular reductions in both right and left dysrhythmia. Conclusions: MMI was associated with improvements across objective and subjective assessments of motor, cognitive, and behavioral control. This proof-of-principle investigation provides preliminary support for the efficacy and feasibility of a novel MMI implemented as part of the school day in an urban school setting with 7-8-year-old children to augment development of at-risk youth. Supplementary Information: The online version contains supplementary material available at 10.1007/s12671-022-02063-7.

Functions of heteromeric and homomeric isoamylase-type starch-debranching enzymes in developing maize endosperm.

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.

Identification of the novel protein QQS as a component of the starch metabolic network in Arabidopsis leaves.

Little is known about the role of proteins that lack primary sequence homology with any known motifs (proteins with unknown functions, PUFs); these comprise more than 10% of all proteins. This paper offers a generalized experimental strategy for identifying the functions of such proteins, particularly in relation to metabolism. Using this strategy, we have identified a novel regulatory function for Arabidopsis locus At3g30720 (which we term QQS for qua-quine starch). QQS expression, revealed through global mRNA profiling, is up-regulated in an Arabidopsis Atss3 mutant that lacks starch synthase III and has increased leaf starch content. Analysis of public microarray data using MetaOmGraph (metnetdb.org), in combination with transgenic Arabidopsis lines containing QQS promoter-GUS transgenes, indicated that QQS expression responds to a variety of developmental/genetic/environmental perturbations. In addition to the increase in the Atss3 mutant, QQS is up-regulated in the carbohydrate mutants mex1 and sis8. A 586 nt sequence for the QQS mRNA was identified by 5' and 3' RACE experiments. The QQS transcript is predicted to encode a protein of 59 amino acids, whose expression was confirmed by immunological Western blot analysis. The QQS gene is recognizable in sequenced Arabidopsis ecotypes, but is not identifiable in any other sequenced species, including the closely related Brassica napus. Transgenic RNA interference lines in which QQS expression is reduced show excess leaf starch content at the end of the illumination phase of a diurnal cycle. Taken together, the data identify QQS as a potential novel regulator of starch biosynthesis.

The complexities of starch biosynthesis in cereal endosperms.

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.

Overlapping functions of the starch synthases SSII and SSIII in amylopectin biosynthesis in Arabidopsis.

BACKGROUND: The biochemical mechanisms that determine the molecular architecture of amylopectin are central in plant biology because they allow long-term storage of reduced carbon. Amylopectin structure imparts the ability to form semi-crystalline starch granules, which in turn provides its glucose storage function. The enzymatic steps of amylopectin biosynthesis resemble those of the soluble polymer glycogen, however, the reasons for amylopectin's architectural distinctions are not clearly understood. The multiplicity of starch biosynthetic enzymes conserved in plants likely is involved. For example, amylopectin chain elongation in plants involves five conserved classes of starch synthase (SS), whereas glycogen biosynthesis typically requires only one class of glycogen synthase. RESULTS: Null mutations were characterized in AtSS2, which codes for SSII, and mutant lines were compared to lines lacking SSIII and to an Atss2, Atss3 double mutant. Loss of SSII did not affect growth rate or starch quantity, but caused increased amylose/amylopectin ratio, increased total amylose, and deficiency in amylopectin chains with degree of polymerization (DP) 12 to DP28. In contrast, loss of both SSII and SSIII caused slower plant growth and dramatically reduced starch content. Extreme deficiency in DP12 to DP28 chains occurred in the double mutant, far more severe than the summed changes in SSII- or SSIII-deficient plants lacking only one of the two enzymes. CONCLUSION: SSII and SSIII have partially redundant functions in determination of amylopectin structure, and these roles cannot be substituted by any other conserved SS, specifically SSI, GBSSI, or SSIV. Even though SSIII is not required for the normal abundance of glucan chains of DP12 to DP18, the enzyme clearly is capable of functioning in production such chains. The role of SSIII in producing these chains cannot be detected simply by analysis of an individual mutation. Competition between different SSs for binding to substrate could in part explain the specific distribution of glucan chains within amylopectin.

Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis.

In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein-protein interactions.

TNF-alpha gene promoter polymorphism at nucleotide -308 and the inflammatory response and oxidative stress induced by cardiac surgery: role of heart failure and medical treatment.

BACKGROUND: Increased TNF-alpha during cardiac surgery is thought to be responsible for perioperative complications. The TNF-alpha gene promoter polymorphism G/A at position -308 has been associated with enhanced TNF-alpha secretion, as has been heart failure. Therefore, the aims of this study were to investigate: (i) whether the TNF-alpha G/A polymorphism is associated with exacerbation of TNF-alpha plasma levels during cardiac surgery; (ii) whether TNF-alpha production is further increased by heart failure and influenced by medical treatment; and (iii) whether this polymorphism is associated with increased oxidative stress and perioperative complications. METHODS: The TNF-alpha gene promoter polymorphism was studied in 100 consecutive patients undergoing cardiac surgery. Of them, 65 were identified with the common allele G/G, whereas 34 patients were with the G/A polymorphism and 1 was A/A. TNF-alpha plasma levels (ELISA) and peroxynitrite content in peripheral blood lymphocytes (flow cytometry) were measured before surgery, before cardiopulmonary bypass (CPB), and 30 min, 4 and 24h after initiation of CPB. RESULTS: The changes observed in TNF-alpha plasma levels during cardiac surgery were unaffected by the G/A polymorphism. TNF-alpha values were elevated before surgery in patients with more advanced NYHA class (1.66+/-0.14, 2.29+/-0.06 and 2.57+/-0.11 ln(mmol/l+1), for NYHA I, II and III; p=0.004) but again they were not correlated with the G/A polymorphism. Peroxynitrite content in lymphocytes was similar upon the initiation of surgery in the G/A and G/G groups and also in all NYHA class groups, and thereafter levels were similarly increased by surgery in all groups. However, analysis of the effect of preoperative medication showed that the mitoK(ATP) channel opener nicorandil reduced TNF-alpha values before surgery and blunted the increase in peroxynitrite caused by surgery. Perioperative complications were not related to either TNF-alpha polymorphism or TNF-alpha and peroxynitrite levels. CONCLUSIONS: The TNF-alpha gene promoter polymorphism G/A at position -308 does not influence TNF-alpha plasma levels during cardiac surgery, is not associated with greater oxidative stress, and does not result in a greater incidence of perioperative complications. However, importantly, treatment with the mitoK(ATP) channel opener nicorandil prior to surgery significantly reduced basal TNF-alpha values and also the oxidative stress induced by surgery.

Starch synthesis in the cereal endosperm.

The pathway of starch synthesis in the cereal endosperm is unique, and requires enzyme isoforms that are not present in other cereal tissues or non-cereal plants. Recent information on the functions of individual enzyme isoforms has provided insight into how the linear chains and branch linkages in cereal starch are synthesized and distributed. Genetic analyses have led to the formulation of models for the roles of de-branching enzymes in cereal starch production, and reveal pleiotropic effects that suggest that certain enzymes may be physically associated. For the first time, tools for global analyses of starch biosynthesis are available for cereal crops, and are heralded by the draft sequence of the rice genome.

Acogimiento residencial terapéutico para niños y adolescentes: una declaración de consenso del Grupo de Trabajo Internacional sobre Acogimiento Residencial Terapéutico / Therapeutic Residential Care for Children and Youth: A Consensus Statement of the International Work Group on Therapeutic Residential Care

En muchos países desarrollados a lo largo del mundo las intervenciones en acogimiento residencial para niños y adolescentes se encuentran en un momento de creciente debate. Ante esta situación, se organizó una cumbre internacional en Inglaterra (primavera de 2016) con expertos de 13 países para reflexionar sobre el acogimiento residencial terapéutico (ART). Se partió de la siguiente definición de ART: "el acogimiento residencial terapéutico implica el uso planificado de un ambiente de convivencia multidimensional, construido a propósito, diseñado para desarrollar o proveer tratamiento, educación, socialización, apoyo y protección a niños y jóvenes con necesidades reconocidas de salud mental o conductuales, en cooperación con sus familias y la colaboración de un amplio espectro recursos comunitarios formales e informales». La reunión se caracterizó por el intercambio de información y evidencias y la preparación de una agenda internacional de investigación. Además, se discutieron las bases para una declaración de consenso. Esta declaración, originalmente publicada en inglés y ahora reproducida en español, comprende, entre otras cuestiones, cinco principios básicos de acogimiento que de acuerdo con el grupo de trabajo en acogimiento residencial terapéutico deben guiar el acogimiento residencial de jóvenes que se preste en todo momento (AU)

In many developed countries around the world residential care interventions for children and adolescents have come under increasing scrutiny. Against this background an international summit was organised in England (spring 2016) with experts from 13 countries to reflect on therapeutic residential care (TRC). The following working definition of TRC was leading: «Therapeutic residential care involves the planful use of a purposefully constructed, multi-dimensional living environment designed to enhance or provide treatment, education, socialization, support, and protection to children and youth with identified mental health or behavioral needs in partnership with their families and in collaboration with a full spectrum of community based formal and informal helping resources». The meeting was characterised by exchange of information and evidence, and by preparing an international research agenda. In addition, the outlines of a consensus statement on TRC were discussed. This statement, originally published in English and now reproduced in a Spanish translation, comprises inter alia five basic principles of care that according to the Work Group on Therapeutic Residental Care should be guiding for residential youth care provided at any time (AU)

Proteins from multiple metabolic pathways associate with starch biosynthetic enzymes in high molecular weight complexes: a model for regulation of carbon allocation in maize amyloplasts.

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes.

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600- and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.

Genome wide co-expression among the starch debranching enzyme genes AtISA1, AtISA2, and AtISA3 in Arabidopsis thaliana.

Each of four starch debranching enzymes (DBE) is distinct and highly conserved across the plant kingdom; however, the specific functions of these proteins in carbohydrate metabolism are not well understood. DBEs function in both biosynthesis and degradation of starch, and two have been shown to function as multimers in various quarternary structures that can contain one or more DBE proteins, i.e. ISA1 homomultimers and ISA1/ISA2 heteromultimers. This study characterizes potential functional relationships between the three isoamylase-type DBE proteins (ISA) of Arabidopsis using a comprehensive bioinformatics analysis and promoter fusion approach to determine tissue-, subcellular-, and temporal specificity of gene expression. The results reveal complementary sets of expression patterns, in particular that AtISA1 (known to be involved in starch biosynthesis) and AtISA2 (a non-catalytic polypeptide) are co-expressed in some conditions in the absence of AtISA3 (known to be involved in starch degradation), whereas in other conditions AtISA2 is co-expressed with AtISA3 in the absence of AtISA1 (AtISA2 and AtISA3, but not AtISA1, are co-expressed specially in root columella cells and leaf hydathodes). Thus, AtISA2 may function in starch degradation, in addition to its role in starch biosynthesis. AtISA3 and several other potential regulatory genes, starch metabolic genes, and transcription factors, are specifically induced during cold acclimation; these transcription factors are candidates for involvement of cold-induced changes in starch metabolism. Finally, bioinformatics analysis using MetaOmGraph (http://www.metnetdb.org/MetNet_MetaOmGraph.htm) identifies Arabidopsis genes of unknown function that might be involved in starch metabolism in the cold.

The maize viviparous15 locus encodes the molybdopterin synthase small subunit.

A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15-specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5' untranslated region as well as a micro-synteny among the cereals.

Mutations affecting starch synthase III in Arabidopsis alter leaf starch structure and increase the rate of starch synthesis.

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.

Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme.

Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes alpha(1-->6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, beta-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.

Mutational analysis of the pullulanase-type debranching enzyme of maize indicates multiple functions in starch metabolism.

Plants contain two types of alpha(1-->6) glucan hydrolase (starch-debranching enzyme [DBE]). Mutations that affect the pullulanase-type DBE have not been described, although defects in isoamylase-type DBE, known in many plant species, indicate a function in starch biosynthesis. We describe a null mutation of a pullulanase-type DBE gene, a Mutator insertion in maize Zpu1. Plants homozygous for the zpu1-204 mutation are impaired in transient and storage starch degradation. Thus, hydrolytic activity of pullulanase-type DBE contributes to starch catabolism. Developing zpu1-204 endosperm accumulates branched maltooligosaccharides not found in the wild type and is deficient in linear maltooligosaccharides, indicating that the pullulanase-type DBE functions in glucan hydrolysis during kernel starch formation. Furthermore, in a background deficient in isoamylase-type DBE, zpu1-204 conditions a significant accumulation of phytoglycogen in the kernel that is not seen in the wild type. Therefore, pullulanase-type DBE partially compensates for the defect in isoamylase-type DBE, suggesting a function during starch synthesis as well as degradation.

Functional consequences of noncognate interactions between CD4+ memory T lymphocytes and the endothelium.

The recruitment of Ag-specific T cells to sites of inflammation is a crucial step in immune surveillance. Although the molecular interactions controlling T cell extravasation are relatively well characterized, the effects of these events on T cell function are still poorly understood. Using an in vitro model of transendothelial migration of human CD4(+) memory T cells, we have investigated the molecular and functional changes induced in T cells that come into contact with the endothelium. First, we show that transendothelial migration is precluded by signals that lead to T cell division. In addition, activation of the transcription factor AP-1, without induction of NF-kappaB, is observed in T cells after noncognate interactions with endothelial cells (EC), a pattern of transcriptional regulation different from that observed in dividing T cells. Up-regulation of certain adhesion (CD11a, CD49d), activation (CD69), and costimulatory (CD86) receptors accompany these transcriptional events. Most importantly, recently migrated T cells display a faster rate of migration when reseeded onto an EC monolayer. Finally, T cells become hyperresponsive to antigenic challenge after noncognate interactions with the endothelium. These effects appear not to be due to the selection of preactivated T lymphocytes, because they occur also in clonal T cell populations and appear to be mediated by alpha(L)beta(2) integrin-CD54 interactions. We conclude that CD4(+) memory T cell extravasation is accompanied by phenotypic and functional changes induced by the interactions with the EC, which favor tissue infiltration by T cells and their further activation once they reach the antigenic site.