Changes in cell wall polysaccharides of green bean pods during development. (33/1703)

The changes in cell wall polysaccharides and selected cell wall-modifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, beta-galactosidase, and alpha-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.  (+info)

Identification of the cDNA encoding human 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway(1). (34/1703)

We report the sequence of a human cDNA encoding a protein homologous to devB (a bacterial gene often found in proximity to the gene encoding glucose-6-phosphate dehydrogenase in bacterial genomes) and to the C-terminal part of human hexose-6-phosphate dehydrogenase. The protein was expressed in Escherichia coli, purified and shown to be 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway. Sequence analysis indicates that bacterial devB proteins, the C-terminal part of hexose-6-phosphate dehydrogenase and yeast Sol1-4 proteins are most likely also 6-phosphogluconolactonases and that these proteins are related to glucosamine-6-phosphate isomerases.  (+info)

cDNA cloning, characterization and stable expression of novel human brain carboxylesterase. (35/1703)

The DNA sequence encoding a novel human brain carboxylesterase (CES) has been determined. The protein is predicted to have 567 amino acids, including conserved motifs, such as GESAGG, GXXXXEFG, and GDHGD which comprise a catalytic triad, and the endoplasmic reticulum retention motif (HXEL-COOH) observed in CES families. Their gene products exhibited hydrolase activity towards temocapril, p-nitrophenyl-acetate and long-chain acyl-CoA. Since the molecular masses of these gene products are similar to those that exist in capillary endothelial cells of the human brain [Yamamda et al. (1994) Brain Res. 658, 163-167], these CES isozymes may function as a blood-brain barrier to protect the central nervous system from ester or amide compounds.  (+info)

Specific hydrolysis of methionyl-tRNA Met f catalyzed by a purified peptide. (36/1703)

A peptide initiation factor purified from rat liver and promoting the binding of initiator tRNA and model initiators to 40S and 80S ribosome at an acid pH liberates methionine and N-acetylmethionine from Trna Met f at neutral reaction. Phenylalanyl-tRNA, N-acetylphenylalanyl-tRNA and methionyl-tRNA Met m are not hydrolyzed under the same conditions. Hydrolysis of methionyl-tRNA Met f is stimulated by the presence of the 40S ribosomal subunit and preceeds at 37 degrees C until all the substrate has been split. No hydrolysis of initiator tRNA or N-acetylmethionyl-tRNA Met f occurs at 0 degrees C. Hydrolysis is slightly stimulated by GTP and MG2+ but not by KCl. The binding and hydrolyzing activity associated with a single protein factor may have an important function in regulating the rate of peptide initiation.  (+info)

A structural view of evolutionary divergence. (37/1703)

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17 degrees C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 A, 1.6 A, and 2.0 A, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the alpha/beta hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.  (+info)

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster. (38/1703)

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.  (+info)

Joint immobilization reduces synovial fluid hyaluronan concentration and is accompanied by changes in the synovial intimal cell populations. (39/1703)

OBJECTIVES: Synovial fluid (SF) of normal joints contains high hyaluronan (HA) concentrations. However, the mechanism by which these are controlled and how they are influenced by articulation and loading are not established. In this study, we have examined whether immobilization influences SF HA concentration and whether this is associated with alterations in the synovial lining. METHODS: Hock joints of five adult sheep were immobilized by external fixation. Twelve weeks later, SF and synovium samples were collected. The HA concentration in SF was assayed using an ELISA-based method. Non-specific esterase (NSE) and uridine diphosphoglucose dehydrogenase (UDPGD) activities were assessed in cryostat sections of snap-chilled synovial samples using cytochemical techniques, and UDPGD activity per cell was measured in synovial lining cells by scanning and integrating microdensitometry. RESULTS: We found that the SF HA concentration was decreased from 1.65+/-0.25 mg/ml in control joints to 0.68+/-0.16 mg/ml in immobilized joints. Synovial intimal cell UDPGD activity decreased from 18.0+/-2.7 U/cell in control joints to 12.2+/-1.5 after immobilization. There was also a decrease in UDPGD-positive intimal cell numbers. Intimal surfaces in controls contained numerous NSE-positive cells, which were rarely observed in intima from immobilized joints. CONCLUSIONS: These results suggest that immobilization decreases SF HA levels and that this is associated with reduced intimal cell UDPGD activity (essential for HA formation). Immobilization also decreased the prevalence of (NSE-positive) intimal macrophages. These findings suggest that mechanosensitive homeostatic mechanisms exist within the synovial intima.  (+info)

Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling. (40/1703)

The Arabidopsis PAD4 gene previously was found to be required for expression of multiple defense responses including camalexin synthesis and PR-1 gene expression in response to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola. This report describes the isolation of PAD4. The predicted PAD4 protein sequence displays similarity to triacyl glycerol lipases and other esterases. The PAD4 transcript was found to accumulate after P. syringae infection or treatment with salicylic acid (SA). PAD4 transcript levels were very low in infected pad4 mutants. Treatment with SA induced expression of PAD4 mRNA in pad4-1, pad4-3, and pad4-4 plants but not in pad4-2 plants. Induction of PAD4 expression by P. syringae was independent of the regulatory factor NPR1 but induction by SA was NPR1-dependent. Taken together with the previous observation that pad4 mutants have a defect in accumulation of SA upon pathogen infection, these results suggest that PAD4 participates in a positive regulatory loop that increases SA levels, thereby activating SA-dependent defense responses.  (+info)