Expression patterns of three α-expansin isoforms in Coffea arabica during fruit development.

As a first step towards understanding the physiological role and regulation of the expansin gene (EXP) family in Coffea arabica fruits during growth and maturation, we identified 11 expansin genes, nine belonging to the α-expansin family (EXPA), one EXLA and one EXLB, through in silico analysis of expressed sequence tags (ESTs). Within the α-expansin family, three isoforms were selected for detailed examination based on their high expression in coffee fruits or because they were specifically induced during different fruit developmental stages, according to the EST information. The expression patterns were analysed in different fruit tissues (perisperm, endosperm and pericarp) of C. arabica cv. IAPAR-59 and C. arabica cv. IAPAR-59 Graúdo, the latter being a closely related cultivar with a larger fruit size. Accumulation of CaEXPA1 and CaEXPA3 transcripts was high in the perisperm (tissue responsible for coffee bean size) and in the early stages of pericarp development. Transcripts of CaEXPA2 were detected only in the pericarp during the later stages of fruit maturation and ripening. There was no detectable transcription of the three EXPs analysed in the endosperm. The observed differences in mRNA expression levels of CaEXPA1 and CaEXP3 in the perisperm of IAPAR-59 and IAPAR-59 Graúdo suggest the participation of these two isoforms in the regulation of grain size.

Genetic differentiation of wild and cultivated populations: diversity of Coffea canephora Pierre in Uganda.

Coffea canephora Pierre ex Frohener is a perennial plant originated from Africa. Two main groups, Guinean and Congolese, have already been identified within this species. They correspond to main refugia in western and central Africa. In this paper we present the analysis of a region that has not yet been studied, Uganda. Two wild, one feral (once cultivated but abandoned for many years), and two cultivated populations of C. canephora from Uganda were evaluated using 24 microsatellite markers. Basic diversity, dissimilarity and genetic distances between individuals, genetic differentiation between populations, and structure within populations were analysed. Expected heterozygosity was high for wild compartments (0.48 to 0.54) and for cultivated and feral ones (0.57 to 0.59), with the number of private alleles ranging from 12 for cultivated genotypes to 37 for a wild compartment. The Ugandan samples show significant population structuring. We compared the Ugandan populations with a representative sample of known genetic diversity groups within the species using 18 markers. Coffea canephora of Ugandan origin was found to be genetically different from previously identified diversity groups, implying that it forms another diversity group within the species. Given its large distribution and extremely recent domestication, C. canephora can be used to understand the effect of refugia colonization on genetic diversity.

[Diagnostic image (358). A neonate with splenomegaly and calcified adrenal glands]. / Diagnose in beeld (358). Een zuigeling met splenomegalie en verkalkte bijnieren.

A 10-day-old male neonate with feeding problems and splenomegaly had bilateral adrenal calcifications on plain abdominal X-ray caused by Wolman disease.

Construction and characterization of a Coffea canephora BAC library to study the organization of sucrose biosynthesis genes.

The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.

[Favourable short-term effects of a multidisciplinary, behavioural therapy, group treatment for overweight or obese children]. / Gunstige kortetermijneffecten van een multidisciplinaire gedragstherapeutische groepsbehandeling voor kinderen met overgewicht of obesitas.

OBJECTIVE: Evaluate the effect of a multidisciplinary, behavioural therapy, group therapy for obese children. DESIGN: Descriptive. METHOD: The treatment consisted of 8 child and 2 parent sessions over a period of 10 weeks, in the Sint Franciscus Gasthuis Hospital in Rotterdam, the Netherlands. Attention was devoted to eating behaviour, physical exercise and psychosocial aspects. Data were collected about children treated in the period 1999-2002. Inclusion criteria were: age 7-14 years, body-mass index (BMI) > or = 25 kg/m2, spoke Dutch, and no problematic behaviour according to the CBCL (total score < 70). Outcome measures were BMI, energy uptake, treadmill endurance time in the Bruce test, and problematic behaviour. There were three measurement points: 3 months before the treatment, immediately before the treatment and immediately after the treatment. RESULTS: 7 of the 78 children who were to participate dropped out. The study cohort consisted of 52 girls and 19 boys with an average age of 10.5 years. In the months prior to the treatment there was no statistically significant change in the outcomes. During the treatment period all of the children lost weight. The mean BMI decreased by 1.7 kg/m2 (-0.3 SDS), the mean daily energy uptake decreased by 1242 kJ and the maximal endurance time increased by 0.8 min (all: p < 0.001). There was no significant decrease in the score for problematic behaviour. CONCLUSION: For the overweight or obese children who followed the treatment programme the BMI and energy intake decreased during the programme and the fitness increased.

Distribution of genomic regions differentiating oak species assessed by QTL detection.

Pedunculate oak and sessile oak are two sympatric interfertile species that exhibit leaf morphological differences. We aimed to detect quantitative trait loci (QTLs) of these traits in order to locate genomic regions involved in species differentiation. A total of 15 leaf morphological traits were assessed in a mixed forest stand composed of Quercus petraea and Q. robur and in a full-sib pedigree of Q. robur. The progeny of the full-sib family were vegetatively propagated in two successive experiments comprising 174 and 216 sibs, and assessments were made on two leaves collected on each of the 1080 and 1530 cuttings corresponding to the two experiments. Traits that exhibited strong species differences in the mixed stand tended also to have higher repeatability values in the mapping population, thus indicating higher genetic control. A genetic map was constructed for QTL detection. Composite interval mapping with the one QTL model was used for QTL detection. From one to three QTLs were detected for 13 traits. In-depth analysis of the QTLs, controlling the five morphological traits that exhibited the highest interspecific differences in the mixed stand, indicated that they were distributed on six linkage groups, with two clusters comprising QTLs of at least two discriminant traits. These results were reinforced when error 1 for QTL detection was set at 5% at the chromosome level, as up to nine clusters could be identified. In conclusion, traits involved in interspecific differentiation of oaks are under polygenic control and widespread in clusters across the genome.

Characterization of a new member of the TNF family expressed on antigen presenting cells.

The TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor superfamily member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

pXen, a utility vector for the expression of GST-fusion proteins in Xenopus laevis oocytes and embryos.

We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

Signaling inositol polyphosphate-5-phosphatase. Characterization of activity and effect of GRB2 association.

An inositol polyphosphate-5-phosphatase (SIP-110) that binds the SH3 domains of the adaptor protein GRB2 was produced in Sf9 cells and characterized. SIP-110 binds to GRB2 in vitro with a stoichiometry of 1 mol of GRB2/0.7 mol of SIP-110. GRB2 binding does not affect enzyme activity implying that GRB2 serves mainly to localize SIP-110 within cells. SIP-110 hydrolyses inositol (Ins)(1,3,4,5)P4 to Ins(1, 3,4)P3. The enzyme does not hydrolyze Ins(1,4,5)P3 that is a substrate for previously described 5-phosphatases nor does it hydrolyze phosphatidylinositol (PtdIns)(4,5)P2. SIP-110 also hydrolyzed PtdIns(3,4,5)P3 to PtdIns(3,4)P2 as did recombinant forms of two other 5-phosphatases designated as inositol polyphosphate-5- phosphatase II, and OCRL (the protein that is mutated in oculocerebrorenal syndrome). The inositol polyphosphate-5-phosphatase enzyme family now is represented by at least 9 distinct genes and includes enzymes that fall into 4 subfamilies based on their activities toward various 5-phosphatase substrates.

A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.

Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2.

BACKGROUND: Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS: The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS: These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Foreign body aspiration in children. The diagnostic value of signs, symptoms and pre-operative examination.

One hundred and fifteen patients, between 6 months and 12 years of age, had bronchoscopy on suspicion of foreign body aspiration. The histories of these patients were studied to examine the diagnostic value of signs, symptoms and examinations, and to determine the time that passed between aspiration and removal of the foreign body. The sensitivity of the symptoms choking and coughing was fairly high (81 and 78%), but the specificity was poor. The sensitivity of a chest radiograph was 82%, the specificity 44%. The sensitivity of radiographs on inspiration and expiration was 80%, the specificity 55%. The patients had been referred with the initial diagnosis foreign body aspiration (80), pneumonia (34), or subglottic laryngitis (1). In 85 patients a foreign body was identified and extracted. The other 30 patients had respiratory tract infections. The initial diagnosis of foreign body aspiration was correct in 61 out of 85 patients. In these cases, the period between aspiration and extraction of the foreign body was a mean 6 days, compared with 55 days, if the initial diagnosis was pneumonia or sub-glottic laryngitis. We conclude that the diagnosis of foreign body aspiration is too often missed, and that, apart from bronchoscopy, diagnostic tools are of little value.

Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphatases.

The question of whether protein tyrosine phosphatases (PTPases) dephosphorylate a multiply phosphorylated peptide in a random or ordered manner was investigated using the synthetic triphosphotyrosyl peptide TRDIY(P)ETDY(P)Y(P)RK, corresponding to the major sites of autophosphorylation of the insulin receptor, as a substrate for four purified PTPases. All four enzymes dephosphorylated the triphospho peptide to produce diphospho, monophospho, and nonphosphorylated forms. Partially dephosphorylated peptides were separated by reverse-phase HPLC, and the di- and monophospho peptides were collected and analyzed by solid-phase sequencing to determine the order of dephosphorylation of the three sites by each of the PTPases. The quantitative analysis of the signals for derivatives of tyrosine and phosphotyrosine generated at positions 5, 9, and 10 of the peptide showed that the low molecular weight human placental PTPase 1B preferentially dephosphorylated the two phosphotyrosines at positions 9 and 10 whereas the integral membrane enzyme CD45 (from human spleen) and the bacterially expressed rat LAR preferentially dephosphorylated the phosphotyrosine at position 5. A second low molecular weight enzyme, termed TCPTPase, did not display any specificity for a particular phosphotyrosyl residue. These results demonstrate that different PTPases exhibit a characteristic pattern of dephosphorylation of the triphospho peptide model substrate, raising the possibility that features in the primary structure surrounding the dephosphorylation site may contribute to substrate specificity.

Active site labeling of a receptor-like protein tyrosine phosphatase.

The inactivation of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase (PTPase), by iodoacetate and not by iodoacetamide suggested that iodoacetate interacts in a highly selective manner with the enzyme. The data indicate that iodoacetate binds at the active site of the enzyme with a stoichiometry of 0.8 mol of iodoacetate bound per mol of rat LAR. A single [14C]iodoacetate-labeled peptide was isolated following endoproteinase Lys-C digestion of the radiolabeled PTPase. Sequence analysis of the active site labeled peptide demonstrates that Cys-1522 contains the radiolabel. This residue has been shown by site-directed mutagenesis to be essential for rat LAR activity (Pot, D. A., Woodford, T. A., Remboutsika, E., Haun, R. S., and Dixon, J. E. (1991) J. Biol. Chem. 266, 19688-19696). Iodoacetate reacts only with the first domain of this double domain PTPase. These results, for the first time, directly identify the highly reactive cysteine residue at the active site of a PTPase and highlight the ability of this residue to participate as a nucleophile in the hydrolysis of phosphate from tyrosine.

Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase.

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.

Forced hydration prior to renography in children with hydronephrosis. An evaluation.

We have developed a method of diuretic renography for the assessment of upper urinary tract obstruction in children. A maximal diuresis under standardised conditions is obtained by forced intravenous hydration over a 2-h period prior to renography. We evaluated the predictive value of this method for the clinical and functional outcome in 73 children with apparent unilateral pelviureteric junction obstruction. The predictive value of a non-obstructive pattern was 94%, while the predictive value of an obstructive pattern could not be assessed accurately because the patients concerned subsequently underwent operation. The method is safe and reliable and offers advantages over conventional diuretic renography.

Purification and characterization of a 43-kDa transcription factor required for rat somatostatin gene expression.

A 43-kDa DNA binding protein which recognizes the TGACGTCA element of the rat somatostatin promoter has been purified from rat brain. Purification of the protein involved initial separation of three sequence-specific binding activities, b1-b3, from each other using DEAE-Sepharose chromatography. The protein corresponding to the b2 complex was further purified to apparent homogeneity by two cycles of sequence-specific DNA affinity chromatography, yielding a single species with an apparent mass of 43,000 daltons on a silver-stained polyacrylamide gel. Sequence-specific DNA binding of this purified protein was demonstrated by Southwestern blotting, renaturation, and DNase I footprinting studies. The 43-kDa protein was phosphorylated on serine residue(s) by the catalytic subunit of cAMP-dependent protein kinase, as shown by phosphoamino acid analysis. Furthermore, the purified protein specifically stimulated transcription from the rat somatostatin promoter in an in vitro transcription system. These results indicate that this 43-kDa protein is a transcription factor required for somatostatin gene expression.

In vitro transcription directed from the somatostatin promoter is dependent upon a purified 43-kDa DNA-binding protein.

In vitro transcription analyses were used to establish the biological function of a 43-kDa affinity-purified DNA-binding protein. The 43-kDa affinity-purified protein protects the region from position -59 to position -35 of the somatostatin promoter from DNase I digestion. This region of the somatostatin promoter harbors the TGACGTCA motif, also found and required for function in a number of other cAMP-responsive and adenovirus E1A-inducible promoters. Efficient and authentic transcription in vitro directed from the somatostatin promoter requires the TGACGTCA promoter element. In vitro transcription assays performed in the presence of somatostatin (positions -60 to -29), enkephalin (positions -105 to -71), and adenovirus type 5 E3 gene (positions -72 to -42) competitor fragments, harboring similar TGACGTCA motifs, selectively inhibit transcription directed from the somatostatin promoter, suggesting that the TGACGTCA element is the site of interaction of a somatostatin gene transactivator. Furthermore, extracts depleted of the TGACGTCA-binding activities by affinity chromatography utilizing a biotinylated oligonucleotide-avidin resin, are incapable of directing transcription from the somatostatin but not from the adenovirus major late promoter. Addition of the purified 43-kDa protein to the affinity-depleted extract restores transcription from the somatostatin promoter. These results are consistent with the 43-kDa protein being a trans-activator of the somatostatin gene.