Biomedical subjects

J Kossmann

Publications and source records attributed to J Kossmann.

28 records · Page 2Linked to original sources

PubMed1997

Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism.

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.

Agrobacterium tumefaciens↗

PubMed1997

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.

Amylopectin↗

PubMed1997

Cloning and characterization of the gene for amylosucrase from Neisseria polysaccharea: production of a linear alpha-1,4-glucan.

The gene for the amylosucrase from Neisseria polysaccharea (ATCC 43768) was cloned by use of a functional expression system in Escherichia coli XL1-Blue. The deduced amino acid sequence of the protein has homology to the sequences of the alpha-amylase class of enzymes, with the highest similarities being found to the sequences of the trehalose synthase from Pimelobacter sp. strain R48 (17) and amylomaltase from Thermotoga maritima (11). However, the regions of highest homology within the alpha-amylase class of enzymes, which are essential for the catalytic activity, are only scarcely found in the sequence of amylosucrase. By using the enzyme isolated from culture supernatants of transformed E. coli cells, it is possible to synthesize linear alpha-1,4-glucans from sucrose, indicating that the enzyme is not capable of producing alpha-1,6-glycosidic linkages on its own.

Amino Acid Sequence↗

PubMed1996

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.).

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.

Amino Acid Sequence↗

PubMed1992

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.

Arachidonic Acids↗

PubMed1991

Cloning and expression analysis of a potato cDNA that encodes branching enzyme: evidence for co-expression of starch biosynthetic genes.

One of the key enzymes involved in the formation of amylopectin, which is the major component of starch, is branching enzyme. A cDNA for potato branching enzyme was cloned by screening a tuber-specific cDNA expression library using an antiserum directed against a denatured preparation of the protein. Complementation of an Escherichia coli strain deficient in branching enzyme was achieved using a construct derived from this clone. Analysis of the expression of the gene in potato revealed a close association with conditions favouring starch biosynthesis. The expression pattern of the gene coding for potato branching enzyme, as analyzed at the mRNA level, closely resembles that of AGPase S, a gene coding for one of the subunits of ADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. This raises the possibility that enzymes involved in the pathway are coordinately regulated at the transcriptional level.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1990

Gene expression during tuber development in potato plants.

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day-length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N-storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP-glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.

Carboxylic Ester Hydrolases↗

PubMed1990

One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose.

The key regulatory step in starch biosynthesis is catalyzed by the tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase). In leaf and storage tissue, the enzyme catalyzes the synthesis of ADP-glucose from glucose-1-phosphate and ATP. Using heterologous probes from maize, two sets (B and S) of cDNA clones encoding potato AGPase were isolated from a tuberspecific cDNA library. Sequence analysis revealed homology to other plant and bacterial sequences. Transcript sizes are 1.9 kb (AGPase B) and 2.1 kb (AGPase S). Northern blot experiments show that the two genes differ in their expression patterns in different organs. Furthermore, one of the genes (AGPase S) is strongly inducible by metabolizable carbohydrates (e.g. sucrose) at the RNA level. The accumulation of AGPase S mRNA was always found to be accompanied by an increase in starch content. This suggests a link between AGPase S expression and the status of a tissue as either a sink for or a source of carbohydrates. By contrast, expression of AGPase B is much less variable under various experimental conditions.

Amino Acid Sequence↗

PubMed1989

Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus.

Two DNA fragments encoding the chromosomal and plasmid copies of the gene (cfxP) encoding phosphoribulokinase (PRK) from the chemoautotrophic bacterium Alcaligenes eutrophus, were sequenced and found to be highly homologous. The gene (cfxF) of another Calvin cycle enzyme, fructose-1,6-bisphosphatase (FBPase), was identified as terminating immediately upstream of cfxP, but was not completely contained on both fragments. A hypothetical, also incompletely contained, open reading frame starts closely downstream from cfxP. Genes cfxF, cfxP, and the third hypothetical gene seem to belong to the same operon. The cfxP genes encode highly homologous PRK isoenzyme subunits consisting of 292 aa residues with calculated Mrs of 33 319 (chromosomal PRKc) and 33 164 (plasmid-encoded PRKp). There is little overall sequence similarity between the bacterial and plant (spinach) PRK, apart from some structural motifs.

Alcaligenes↗

PubMed1980

Effects of cryptand 2.2.1Py on the fast sodium current in cardiac Purkinje fibers.

For study of dose-dependent influence on sodium current, sheep heart Purkinje fibers were perfused with a cryptand (2.2.1Py) and measured for resting potential, overshoot, amplitude and duration of action potential, refractory period, and rate of rise. Concomitant investigation was made by voltage clamp. The cryptand was found to depress maximal rate rise with increase of concentration and induce shift toward negative potential with increase of dose. Also, effect on sodium current kinetics was observed but not on resting potential. The findings indicate that 2.2.1Py action probably is not primarily ionophores, negating the idea that cryptands promote transmembrane ion exchange.

Action Potentials↗