Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

Insecticidal activity of the protein encoded by the cryV gene of Bacillus thuringiensis kurstaki INA-02.

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.

Quantitative fecal carbohydrate excretion in premature infants.

Despite relative lactase deficiency and pancreatic insufficiency, premature infants are fed formulas containing 50% lactose plus 50% glucose polymers. We measured total fecal carbohydrate excretion in six healthy 32-wk gestation premature infants who had been fed two 0.784-kcal/g formulas that were similar except for the carbohydrate source (100% lactose vs 50% lactose plus 50% glucose polymers). Using a cross-over design with the first formula randomly assigned, two 72-h balance studies were performed with carmine red, an intermittent external marker, and polyethylene glycol (PEG), a continuous internal marker. Formula and stools were analyzed for total carbohydrate (anthrone method) and PEG. There were no significant differences between the two formula periods for carbohydrate intake, mean daily stool output, or fecal carbohydrate excretion. Mean fecal carbohydrate excretion was less than 0.2 g/d, or less than 1% of carbohydrate intake. Thus, older (32-wk gestation) premature infants fed either 100% lactose or 50% lactose plus 50% glucose polymers have minimal fecal losses of intact carbohydrate.

Absorption of food protein antigen in infants with food protein-induced enterocolitis.

Increased gastrointestinal absorption of intact antigen with systemic immunization has been considered a major etiologic factor in the development of food sensitivity. We attempted to test this hypothesis in infants with suspected food protein-induced entercolitis by measuring serum ovalbumin (OVA) concentrations after ingestion of egg white (prior to the performance of good challenges to establish this diagnosis). We first noted significant underestimation of serum OVA concentrations in the presence of even low serum anti-OVA antibody concentrations (greater than 1:12). Next, using selected noninhibitory sera, we found that all infants studied absorbed some OVA, there was no correlation between serum OVA levels and age (3-11 months), and there was no significant difference between serum OVA concentrations in infants who subsequently had positive oral food challenge responses (120 +/- 67 ng/ml) and a matched group with negative challenges (102 +/- 80). These data do not support the hypothesis that "intestinal closure" (antigen exclusion) occurs in the neonatal period or the role of antigen absorption as the major etiological factor in the development of food sensitivity. Better methods of quantitating macromolecular absorption must be developed before the role of antigen absorption in food sensitivity can be assessed. Of note, urinary excretion of intact OVA also occurred. This varied greatly from one voiding to the next and continuing for at least 13 hr after ingestion.

Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase.

An indispensable part of the hydrogen-recycling system in Bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kDa subunits. The gene encoding the large subunit is located on a 5.9-kilobase fragment of the H2-uptake-complementing cosmid pHU52 [Zuber, M., Harker, A.R., Sultana, M.A. & Evans, H.J. (1986) Proc. Natl. Acad. Sci. USA 83, 7668-7672]. We have now determined that the structural genes for both subunits are present on this fragment. Two open reading frames are present that correspond in size and deduced amino acid sequence to the hydrogenase subunits, except that the small-subunit coding region contains a leader peptide of 46 amino acids. The two genes are separated by a 32-nucleotide intergenic region and likely constitute an operon. Comparison of the deduced amino acid sequences of the B. japonicum genes with those from Desulfovibrio gigas, Desulfovibrio baculatus, and Rhodobacter capsulatus indicates significant sequence identity.

Inducible expression of cytokinin biosynthesis in Agrobacterium tumefaciens by plant phenolics.

Nopaline strains of Agrobacterium tumefaciens contain a gene, tzs, that encodes a cytokinin biosynthetic prenyl transferase. The gene is located adjacent to the Ti plasmid virulence region and is constitutively expressed at low levels. As a result, bacteria containing tzs secrete low levels of zeatin into the medium. We find zeatin secretion to be induced more than 100-fold by acetosyringone, one of a number of naturally occurring phenolics produced by plants in response to wounding. Induction was very sensitive to the pH of the medium (optimum pH 5.5) and was due to massive overexpression of tzs-encoded cytokinin prenyl transferase activity. The relative ability of members of a set of phenols to induce tzs expression was examined and found to be parallel to that reported for activation of other virulence genes. A series of molecular cloning experiments established that virA and virG, two genes known to be essential to the virulence induction process, were necessary and sufficient for phenolic-induced tzs expression. Sequences present in the promoter region of tzs were found to be similar to those present in genes regulated by bacterial two-component positive regulatory systems.

4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography.

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.

Support for a conservative approach to mixed intrahepatic and extrahepatic biliary hypoplasia.

This report describes the pitfalls in the diagnosis of extrahepatic biliary atresia in an infant with paucity of the interlobular bile ducts.

Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58.

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two-component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure-function model for the gene product are discussed.

Carbohydrate malabsorption is minimal in school-age cystic fibrosis children.

Fifteen school-age cystic fibrosis children, participating in a year-long nutritional management study, were hospitalized at six-month intervals for balance studies during which they continued "free-choice" diets and their usual enzyme supplementation. Stools were analyzed for fat and protein by conventional methods and for carbohydrate using a recently validated anthrone method. Despite persistent fat and protein malabsorption, less than 1% of ingested carbohydrate was lost intact in the stools. Comparison of baseline and placebo balance studies showed fecal excretion of carbohydrate to be independent of intake, in contrast to the fat and protein results. Using a thin-layer chromatography method capable of detecting microgram quantities of urinary organic acids, no short-chain fatty acids were detected in the stool. Further exploration of carbohydrate as a dietary energy source for this patient group with increased energy demands should be pursued.

Quantitation of fecal carbohydrate excretion in patients with short bowel syndrome.

We performed inpatient balance studies in 11 patients to evaluate the role of carbohydrate malabsorption in the pathogenesis of the diarrhea seen in short bowel syndrome. Stool weight, total reducing substance as measured by Clinitest, and total fecal carbohydrate as measured by anthrone were determined. Patients had markedly increased fecal carbohydrate excretion, up to 65% of dietary carbohydrate intake. When the diet contained oligosaccharides, measures of total reducing substance greatly underestimated fecal carbohydrate excretion and were unreliable for quantitation. Stool weight correlated with total fecal carbohydrate excretion and with total reducing substance (r = 0.79, p less than 0.001). Multiple balance studies in 2 patients suggested a relationship between both the amount and type of dietary carbohydrate and fecal carbohydrate excretion. These studies suggest that carbohydrate malabsorption is a major cause of the watery diarrhea and subsequent fluid, electrolyte, and acid-base imbalance seen in patients with short bowel syndrome.

Secretion of Zeatin, Ribosylzeatin, and Ribosyl-1'' -Methylzeatin by Pseudomonas savastanoi: Plasmid-Coded Cytokinin Biosynthesis.

Cytokinin production by strains of the phytopathogenic bacterium Pseudomonas syringae pv savastanoi was measured by immunoaffinity chromatography of the culture medium on immobilized anti-cytokinin antibodies, followed by high performance liquid chromatography, radioimmunoassay and mass spectrometry. P. savastanoi strain PB213-2 secretes zeatin (80 nanograms per milliliter) and ribosylzeatin (80 nanograms per milliliter). Even higher levels of zeatin (400 nanograms per milliliter) are produced by the olive-specific strain EW1006, which also produces 180 nanograms per milliliter of the recently identified cytokinin, ribosyl-1'' -methylzeatin. The amounts secreted were approximately 1000 times greater than those secreted by Agrobacterium tumefaciens (DA Regier, RO Morris 1982 Biochem Biophys Res Commun 104: 1560-1566). Examination of cytokinin production by plasmid deletion mutants of PB213-2 and EW1006 indicated that cytokinin biosynthesis was specified, at least in part, by plasmid-borne genes. A fragment of the 105 kilobase pair plasmid from EW1006 was cloned into Escherichia coli where its expression resulted in dimethylallyl transferase activity and the secretion of zeatin.

Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci.

The nucleotide sequence of a Pseudomonas trans-zeatin producing gene (ptz) from the pCK1 plasmid of Pseudomonas syringae pv. savastanoi strain 1006 has been determined. This gene confers upon E. coli the ability to synthesize and secrete several cytokinins including trans-zeatin, iso-pentenyladenine and their respective N9-ribosyl derivatives. Sequence analysis indicates an open reading frame encoding a protein of 234 amino acids with a molecular weight of 26,816. Significant sequence homology is found between ptz and both the tzs and tmr genes from Agrobacterium tumefaciens. The results suggest a close relationship between the cytokinin biosynthetic pathways in P. savastanoi and A. tumefaciens.

A simple spectrophotometric method for quantitative fecal carbohydrate measurement.

A simple quantitative assay was developed for measuring total fecal carbohydrate (CHO) excretion using stools obtained during balance studies performed for fecal fat determination. This spectrophotometric method utilizes Dreywood's anthrone reagent which reacts with equal weights of CHO whether monosaccharide or polysaccharide. Analysis of known dietary CHO solutions yielded greater than 90% of the known theoretical concentration. Recovery of CHO (Polycose) added to fresh stool was greater than 95%, inter-assay coefficient of variation (CV) 6.2%. Stool specimens stored frozen and analyzed over a 21-month period yielded stable results. Fecal CHO excretion/day determined in 32 normal patients, ages 1 month to 13 years, on diets with varying CHO sources, ranged from 0.04 to 0.85 g, average 0.33 g, SD +/- 0.24. Three patients with diseases known to be associated with CHO malabsorption studied showed markedly increased total fecal CHO excretion, up to 53% of their CHO intake. Quantitative fecal CHO excretion in these patients allowed for assessment of the severity of their disease and provided a means for evaluating the use of different dietary CHO sources in their management.

Milk- and soy protein-induced enterocolitis: evidence for lymphocyte sensitization to specific food proteins.

Stimulation ( [3H]thymidine incorporation) of blood lymphocytes cultured with food proteins was evaluated in infants with food protein-induced enterocolitis and correlated with the results of oral diagnostic challenges with the same foods (soy, cow's milk, and egg white). The geometric mean stimulation index for lymphocytes from patients with positive oral soy protein challenge that were cultured with soy protein was 8.5, and for patients with positive cow's milk challenge the stimulation index was 6.0 when casein was used in the cultures. Both values are significantly different from the values obtained from patients with negative oral challenges (p less than 0.01). The enhanced lymphocyte responses were specific for the food proteins responsible for clinical symptoms. It is not clear whether these lymphocyte responses are due to systemic immunization secondary to macromolecular absorption, or to an abnormality in immune regulation such as a delay in the development of oral tolerance mechanisms. They suggest, however, that circulating lymphocytes sensitive to the food antigens that produce the clinical symptoms are frequent in infants with this discrete form of food protein hypersensitivity.

Food protein-induced enterocolitis: altered antibody response to ingested antigen.

To evaluate the role of immunologic mechanisms in one specific syndrome of food intolerance in infants, food protein-induced enterocolitis, we measured class-specific serum antibodies to three food proteins, ovalbumin, soy, and cow milk, prior to diagnostic food challenges in 18 infants suspected to have this syndrome. Infants with positive challenge reactions to egg, soy, or cow milk had 5-10 times higher levels of IgA antibody directed against that food than did the infants with negative challenges. Levels of IgG antibody to soy and egg were also significantly higher (greater than 10-fold) in infants with positive challenge responses. There was no significant difference in levels of IgM food antibodies between the two groups. IgA anti-soy antibody levels rose in all 12 infants tested 2-10 weeks after a single soy feeding (challenge). However, IgM anti-soy antibody increased in the five infants who had a negative response to the challenge feeding and decreased in those seven with a positive response. The difference between the two groups was statistically significant (P less than 0.01). Some correlation existed (r = -0.68) between the increase in IgA anti-soy antibody and the decrease in IgM anti-soy antibody for infants with positive soy challenges. Although a pathogenic role for these antibodies is not proven, the findings suggest an altered immunologic response to ingestion of food antigens in infants with food protein-induced enterocolitis.

Purification and properties of a 4-methylene-L-glutamine amidohydrolase from peanut leaves.

A 4-methylene-L-glutamine amidohydrolase has been partially purified from leaf extracts of 2-week germinated peanuts (Arachis hypogaea). Purification steps include DEAE-Sephacel and gel filtration chromatography as well as chromatofocusing; amidohydrolase purified 300- to 400-fold is obtained. The enzyme has an approximate molecular weight of 45,000 as determined by gel filtration chromatography and polyacrylamide gel electrophoresis, a broad activity optimum between pH 8.0 and 9.0, and is highly specific toward 4-methylene-L-glutamine. Of a number of amides tested as substrate, only L-glutamine serves 20% as effectively as the methylene-substituted analog. Multiple bands of activity, seen when enzyme samples are electrophoresed on polyacrylamide gels, are not completely resolved but appear to be very similar in molecular weight, pK values, and subcellular localization. Activity is not affected either by added thiols, metal ions, sulfhydryl-reacting reagents, or metal-ion chelators; it is inhibited by borate ions but stimulated by sodium dodecyl sulfate. This amidohydrolase activity is absent in imbibed seeds, but on germination increases rapidly in the cotyledons and leaves for 3 weeks followed by a gradual decline as the plant matures. Activity is almost completely (95%) localized in the leaves and cotyledons. Differential centrifugation studies indicate that the enzyme is found solely in the soluble fraction of peanut leaves.