Biomedical subjects

C Benning

Publications and source records attributed to C Benning.

At least 37 records · Page 2Linked to original sources

PubMed1999

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearance, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thaliana tt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds.

Arabidopsis↗

PubMed1999

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase.

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.

Journal Article↗

PubMed1999

Characterization of skin cytokines in bullous pemphigoid and pemphigus vulgaris.

The purpose of this study was to determine cytokine and cell marker expression in perilesional skin biopsies from patients with the autoimmune blistering diseases bullous pemphigoid (BP, n = 21) and pemphigus vulgaris (PV, n = 7). Immunohistochemistry and in situ hybridization were used to detect T helper (Th)1 [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-5, IL-13) protein and mRNA. Perilesional skin biopsies from patients with BP were characterized by the deposition of IL-4, IL-13 and IL-5. In patients with BP, IL-4 and IL-13 localized to mononuclear cells within the dermal infiltrate while IL-5 was predominately expressed at the dermal-epidermal junction. BP skin sections also expressed vascular cell adhesion molecule 1 on endothelial cells, not seen in patients with PV. PV biopsies were remarkable for a mixed Th1/Th2 pattern of cytokine expression, including the presence of IL-2, IFN-gamma and IL-4 and the absence of IL-5 and IL-13. In situ hybridization detected mRNA for IL-4 and IL-5 in the cellular infiltrate of BP patients, and IL-2 in a patient with PV. In vitro binding assays demonstrated that normal human eosinophils, activated by coculture in IL-5, bound preferentially to BP skin sections that contained detectable in vivo bound IL-5. The predominance of Th2 cytokines in BP, in association with increased binding of eosinophils in vitro, suggests that Th2 cytokines are relevant in the recruitment and adhesion of eosinophils within the dermal infiltrates of patients with BP, and may play a part in the pathogenesis of blister formation.

Biomarkers↗

PubMed1999

['Microbial air pollutants'. Emission sources and preventive measures].

Already in the planning or monitoring phase, measures have to be realized for the reduction in emissions of bio-aerosols in biological waste treatment plants. For this purpose, operation-related measures (in open and partially closed plants) and technology-related measures (in closed plants with automatic ventilation) can be taken. A survey on the currently feasible measures for reduction is submitted. The realization has to be adapted to the individual site and type of plant. An enormous demand for examinations is envisaged in order to enable a qualified assessment of the emission behaviour of diverse waste air systems or process steps.

Air Microbiology↗

PubMed1998

The phospholipid-deficient pho1 mutant of Arabidopsis thaliana is affected in the organization, but not in the light acclimation, of the thylakoid membrane.

The pho1 mutant of Arabidopsis has been shown to respond to the phosphate deficiency in the leaves by decreasing the amount of phosphatidylglycerol (PG). PG is thought to be of crucial importance for the organization and function of the thylakoid membrane. This prompted us to ask what the consequences of the PG deficiency may be in the pho1 mutant when grown under low or high light. While in the wild-type, the lipid pattern was almost insensitive to changes in the growth light, PG was reduced to 45% under low light in the mutant, and it decreased further to 35% under high light. Concomitantly, sulfoquinovosyl diacylglycerol (SQDG) and to a lesser extent digalactosyl diacylglycerol (DGDG) increased. The SQDG increase correlated with increased amounts of the SQD1 protein, an indicator for an actively mediated process. Despite of alterations in the ultrastructure, mutant thylakoids showed virtually no effects on photosynthetic electron transfer, O2 evolution and excitation energy allocation to the reaction centers. Our results support the idea that PG deficiency can at least partially be compensated for by the anionic lipid SQDG and the not charged lipid DGDG. This seems to be an important strategy to maintain an optimal thylakoid lipid milieu for vital processes, such as photosynthesis, under a restricted phosphate availability.

Arabidopsis↗

PubMed1998

Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana.

Photosynthetic membranes of higher plants contain specific nonphosphorous lipids like the sulfolipid sulfoquinovosyl diacylglycerol in addition to the ubiquitous phospholipid phosphatidylglycerol. In bacteria, an environmental factor that drastically affects thylakoid lipid composition appears to be the availability of phosphate. Accordingly, we discovered an increase in the relative amount of sulfolipid and a concomitant decrease in phosphatidylglycerol in Arabidopsis thaliana grown on medium with reduced amounts of phosphate, as well as in the pho1 mutant of A. thaliana deficient in phosphate transport. To investigate the molecular basis of the observed change in lipid composition, we isolated a cDNA of A. thaliana, designated SQD1, that encodes a protein involved in sulfolipid biosynthesis as suggested by three lines of evidence. First, the cDNA shows high sequence similarity to bacterial sqdB genes known to be essential for sulfolipid biosynthesis; second, the SQD1 gene product is imported into chloroplasts where sulfolipid biosynthesis takes place; and third, transgenic plants expressing SQD1 in antisense orientation show a reduction in sulfolipid content. In the pho1 mutant as well as in wild-type plants grown under reduced phosphate availability, increased amounts of SQD1 mRNA and SQD1 protein are detected, suggesting that the increase in sulfolipid content under phosphate limitation is the result of an increased expression of at least one gene required for sulfolipid biosynthesis in A. thaliana. It is suggested that a certain amount of anionic thylakoid lipid is maintained by substituting sulfolipid for phosphatidylglycerol under reduced phosphate availability.

Amino Acid Sequence↗

PubMed1998

AGO1 defines a novel locus of Arabidopsis controlling leaf development.

An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA resulted in a partial mutant phenotype. Sense expression caused some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms.

Amino Acid Sequence↗

PubMed1998

The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis.

Uridine 5'-diphospho-glucose-4-epimerase (UDP-Glc epimerase) catalyses the reversible epimerization of UDP-galactose and UDP-glucose. In contrast to bacteria and yeast, expression of the UDP-Glc epimerase gene in Arabidopsis was found not to be induced by galactose. To elucidate the metabolic role of this enzyme, transgenic Arabidopsis plants expressing the respective cDNA in sense or antisense orientation were constructed, leading to a range of plant lines with different UDP-Glc epimerase activities. No alterations in morphology were observed and the relative amounts of different galactose-containing compounds were not affected if the plants were raised on soil. However, on agar plates in the presence of galactose, the growth of different lines was increasingly repressed with decreasing enzyme activity, and an increase in the UDP-Gal content was observed in parallel, whereas the UDP-Glc content was nearly constant. The amount of galactose in the cell wall was increased in plants with low UDP-Glc epimerase activity grown on galactose, whereas the cellulose content in the leaves was not altered. Furthermore, starch determined at different times of the day was highly abundant in plants with low UDP-Glc epimerase activity in the presence of galactose. It is proposed that low endogenous UDP-Glc epimerase activity is responsible for the galactose toxicity of the wild-type. Possible mechanisms by which the starch content might be modulated are discussed.

Arabidopsis↗

PubMed1998

wrinkled1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism.

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

Arabidopsis↗

PubMed1997

Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol.

The primary biochemical defect in the genetically well characterized dgd1 mutant of Arabidopsis thaliana causes a 90% reduction in the relative amount of the galactolipid digalactosyldiacylglycerol (DGDG). To study the effect of this DGDG deficiency on photosystem II (PS II), time-resolved transients of laser-flash-induced changes of the relative fluorescence quantum yield Fvar,rel(t) were measured in whole leaves from wild-type and the dgd1 mutant. The results obtained reveal (i) in untreated leaves the decay kinetics of Fvar, rel(t) reflecting QA.- reoxidation by endogenous plastoquinone are very similar in wild-type and the dgd1 mutant at room temperature, (ii) the Arrhenius plot of the temperature dependence of electron transfer from QA.- to QB exhibits a break point at about 19 degrees C in wild-type and about 12 degrees C in the dgd1 mutant, (iii) in leaves treated with DCMU the slow reoxidation of QA.- by the PS II donor side is blocked to a much higher extent in the dgd1 mutant (about 50%) compared to wild-type (about 10%), and iv) the normalized amplitude of Fvar,rel(t = 1 micros) reflecting the percentage of fast P680.+ reduction by YZ exhibits a characteristic period four oscillation in wild-type while this feature is strongly damped in the dgd1 mutant. Presumably, the severe DGDG deficiency is causing the thermal down shift of a lipid phase transition that affects the QA.- reoxidation by QB. Most strikingly, the properties of the WOC are modified as a result of reduced DGDG content. Thus, the lipid DGDG appears to be of structural relevance for the WOC.

Arabidopsis↗

PubMed1997

Accumulation of sulfoquinovosyl-1-O-dihydroxyacetone in a sulfolipid-deficient mutant of Rhodobacter sphaeroides inactivated in sqdC.

The biosynthesis of the sulfolipid sulfoquinovosyl diacylglycerol in the purple bacterium Rhodobacter sphaeroides requires at least four genes:sqdA, sqdB, sqdC, and sqdD. As part of our strategy aimed at the elucidation of the function of the different sqd gene products, we insertionally inactivated sqdC of R. sphaeroides. The resulting sqdC null mutant showed only a 90% reduction in sulfolipid content. Apparently, the sqdC gene product is required for optimal sulfolipid biosynthesis, but either catalyzes no essential reaction in the pathway or can be functionally replaced to a certain extent by a different protein. The mutant accumulated a 35S-labeled compound that was purified to homogeneity from cell extracts. Matrix-assisted laser desorption mass spectrometry and nuclear magnetic resonance spectroscopy provided conclusive structural evidence to identify the compound as alpha-D-sulfoquinovosyl-1-O-dihydroxyacetone that exists in two interconvertible, keto and hemiacetal forms. Incubation of wild-type protein extracts with the labeled compound did not result in the incorporation into sulfolipid as would be expected for an intermediate of the pathway. Based on our results we propose that the sqdC gene product mediates the substrate specificity of the UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase that is encoded by sqdD and that catalyzes the final reaction of sulfolipid biosynthesis.

Bacterial Proteins↗

PubMed1997

Changes in the composition of the photosynthetic apparatus in the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana.

The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.

Arabidopsis↗

PubMed1996

A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyl diacylglycerol.

The sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol is associated with the thylakoid membranes of many photosynthetic organisms. Previously, genes involved in sulfolipid biosynthesis have been characterized only in the purple bacterium Rhodobacter sphaeroides. Unlike plants and cyanobacteria, photosynthesis in this bacterium is anoxygenic due to the lack of a water splitting photosystem II. To test the function of sulfolipid in an organism with oxygenic photosynthesis, we isolated and inactivated a sulfolipid gene of the cyanobacterium Synechococcus sp. PCC7942. Extensive analysis of the sulfolipid-deficient null mutant revealed subtle changes in photosynthesis related biochemistry of O2. In addition, a slight increase in the variable room temperature chlorophyll fluorescence yield was observed. Regardless of these changes, it seems unlikely that sulfolipid is an essential constituent of a functional competent water oxidase or the core antenna complex of photosystem II. However, reduced growth of the mutant under phosphate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids under phosphate-limiting growth conditions.

Amino Acid Sequence↗

PubMed1996

Functional expression of uridine 5'-diphospho-glucose 4-epimerase (EC 5.1.3.2) from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli.

It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants. Searching a database of expressed sequence tags, a cDNA from Arabidopsis thaliana (clone 108G20T7) with sequence similarity to UDP-glucose epimerase was identified and further analyzed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein sequence showed a high degree of similarity to UDP-glucose epimerase sequences from bacteria, rat, and yeast. Complementation of the Saccharomyces cerevisiae gal1O mutant and expression of an active enzyme in Escherichia coli demonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It showed a broad pH optimum of 7.0 to 9.5 and a Km of 0.11 mM. The UDP-glucose epimerase activity was not dependent on the addition of the cofactor NAD+ and was only moderately inhibited by high salt concentrations. Tissue-specific Northern analysis showed that the gene is expressed in all tissues of A. thaliana with highest expression levels in the stems and roots. Based on Southern analysis, there seems to be a single gene encoding UDP-glucose epimerase in A. thaliana. The cDNA analyzed during this study is the first known to encode a sugar-nucleotide modifying enzyme from higher plants. Its availability provides the means to investigate the role of UDP-glucose epimerase for the biosynthesis of UDP-galactose as precursor of galactolipids and cell wall polysaccharides.

Amino Acid Sequence↗

PubMed1995

Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

The sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol is found in the photosynthetic membranes of all plants and most photosynthetic bacteria. Progress toward the elucidation of the pathway for sulfolipid biosynthesis has been slow in the past. However, the recent isolation of three genes of the photosynthetic bacterium Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading frame predicted to encode a protein with amino acid sequence similarity to sugar nucleotide-dependent glycosyltransferases. The UDP-sulfoquinovose:diacylglcerol sulfoquinovosyltransferase thought to catalyze the last step of sulfolipid biosynthesis belongs to this group of glycosyltransferases. To test whether this open reading frame encodes the sulfoquinovosyltransferase of R. sphaeroides, it was inactivated by gene replacement avoiding polar mutagenesis. The resulting sulfolipid-deficient mutant defines a new gene, designated sqdD. Mutant cells grown in the presence of [35S]sulfate accumulate a water-soluble 35S-labeled compound. The purified compound was tentatively identified by co-chromatography with standards and enzymatic conversion as UDP-sulfoquinovose, the final precursor of sulfolipid biosynthesis. This result strongly suggests that the inactivation of sqdD causes a metabolic block in the last step of sulfolipid biosynthesis.

Bacterial Proteins↗

PubMed1995

Accumulation of a novel glycolipid and a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.

Betaine↗

PubMed1995

Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol.

The galactolipids monogalactosyl and digalactosyl diacylglycerol occur in all higher plants and are the predominant lipid components of chloroplast membranes. They are thought to be of major importance to chloroplast morphology and physiology, although direct experimental evidence is still lacking. The enzymes responsible for final assembly of galactolipids are associated with the envelope membranes of plastids, and their biochemical analysis has been notoriously difficult. Therefore, we have chosen a genetic approach to study the biosynthesis and function of galactolipids in higher plants. We isolated a mutant of Arabidopsis that is deficient in digalactosyl diacylglycerol by directly screening a mutagenized M2 population for individuals with altered leaf lipid composition. This mutant carries a recessive nuclear mutation at a single locus designated dgd1. Backcrossed mutants show stunted growth, pale green leaf color, reduced photosynthetic capability, and altered thylakoid membrane ultrastructure.

Arabidopsis↗

PubMed1993

Isolation and functional expression in Escherichia coli of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides.

Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine. In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase. Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine. A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants. Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine. A protein extract derived from the E. coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine. Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R. sphaeroides.

Amino Acid Sequence↗