Effects of immobilization on the kinetics of enzyme-catalyzed reactions. II. Urease in a packed-column differential reactor system.

Urease from Jack bean was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column differential reactor. To facilitate comparison, the urease was immobilized by both diazo and glutaraldehyde coupling. The kinetic properties of immobilized urease were similar to those of the soluble enzyme and different immobilization methods did not appreciably alter the kinetic properties. The affects of three different amino acid activators appear to follow predictions obtained from a relatively simple competitive model, except at very low substrate levels.     

Divergent effects of adrenocorticotropin and melanotropin on isolated rat and rabbit adipocytes.

The stimulation of lipolysis in isolated rat and rabbit fat cells by adrenocorticotropin (ACTH) and alpha-melanotropin has been studied. The concentration of alpha-melanotropin required for half maximal stimulation is 0.23 times that of ACTH in rabbit adipocytes but as high as 1140 times that of ACTH in rat fat cells. Chemical modification of the tryptophan residue in ACTH and melanotropin resulted in a loss of lipolytic activity in rat adipocytes and an increase in lipolytic potency in rabbit fat cells. These differences between rat and rabbit fat cells were evident when stimulation of cyclic AMP synthesis was measured in isolated cells or ghosts. The results are discussed in terms of the difference in the hormone receptors of the fat cells of the two species.     

Inhibition of DNA synthesis in adrenocortical cells by cytochalasin B

ACTH inhibits DNA synthesis in normal rat and mouse tumor Y-1 adrenocortical cells within the same concentration range that it stimulates steroidogenesis. These processes can be independently regulated as demonstrated by the divergent actions of cytochalasin B on these cells. In the normal cells, cytochalasin B does not increase steroidogenesis in serum-free or serum-containing media, and it decreases the stimulation produced by ACTH. In the absence of serum, the Y-1 cells respond in a similar way. However, in serum-containing media, cytochalasin B increases steroidogenesis in these cells and does not inhibit the response to ACTH. In both cell types, cytochalasin B inhibits [3H]thymidine incorporation into DNA by a mechanism different than that of ACTH. In the Y-1 cells, this inhibition is caused by a decreased uptake of [3H]thymidine into the cell, which probably reflects a decreased transport across the cell membrane. In the normal cells, cytochalasin B, like ACTH, does not affect [3H]thymidine transport, but it decreases DNA synthesis much more rapidly than does ACTH. This inhibition may be the result of the disruption of microfilaments by cytochalasinB, because our evidence indicates that it is not caused by a decrease in glucose uptake by the cells.     

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.     

Population abundance and movement of Frankliniella species and Orius insidiosus in field pepper

1 A recent study revealed the capacity of the Orius insidiosus to suppress populations of Frankliniella spp. in field pepper during the spring when thrips are rapidly colonizing and reproducing. In this study, population abundance in pepper during spring, summer, and autumn was determined to understand better predator/prey dynamics under local conditions. Local movement between pepper flowers also was quantified to examine how population attributes of the predator allow suppression of rapidly moving populations of prey. 2 Randomized complete block experiments established in the autumn of 1998 and the spring of 1999 included treatments of biological and synthetic insecticides, which altered the population densities of predator and prey. Numbers of O. insidiosus in relation to prey were sufficient in 1998 to prevent build‐up of thrips populations. In 1999, populations of thrips were unable to recover from near extinction owing to persistence of the predator. The predator rapidly recolonized plots treated with insecticide. 3 Greenhouse plants of the same age as field plants were used to monitor movement by predators and prey. Movement by F. occidentalis was limited, whereas F. tritici and F. bispinosa moved rapidly to the greenhouse plants. The males of each thrips species moved more rapidly than the females. There was evidence that rapid movement assisted F. tritici and F. bispinosa in avoiding predation, but O. insidiosus also moved very rapidly to the greenhouse plants. This attribute explains the predator's ability to suppress thrips rapidly even when populations are rapidly colonizing and reproducing in the flowers.     

Limited proteolysis and active-site labeling studies of soybean lipoxygenase.

Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an N-terminal fragment of about 60 kDa. Under conditions that led to complete cleavage of the protein as judged by SDS-polyacrylamide gel electrophoresis, the catalytic activity of the protein was either reduced slightly (chymotrypsin) or enhanced (trypsin). The characteristics of the cleaved enzymes were the same as for native lipoxygenase 1 in all aspects examined: insensitivity to cyanide, fluoride, and EDTA, regiochemical and stereochemical consequences of catalysis, and EPR spectroscopy upon oxidation by product. The two fragments apparently were tightly associated as they could not be resolved under conditions which preserved the catalytic activity. Both native and protease-cleaved lipoxygenase 1 formed covalent adducts when treated with either 14C-phenylhydrazine or 4-nitrophenylhydrazine. The label was found only in the 60-kDa fragment and following complete trypsin digestion was associated with a peptide beginning after Lys-482 in the primary sequence. Therefore labeling occurred in the vicinity of the conserved histidine cluster which has been postulated as the iron-binding site. From these observations it appears that lipoxygenase 1 exists as a pair of tightly associated domains with the iron-binding site located in the larger of the two.     

Next-generation high-density self-assembling functional protein arrays

We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.     

Hypoglycemic Effect of Macrocyclic Binuclear Oxovanadium (IV) Complex on Streptozotocin-Induced Diabetic Rats

Though vanadium complexes mimic the action of insulin, owing to their toxicity, research is still in progress for a new vanadium complex with maximum efficacy at low concentration and without any side effects. A novel macrocyclic binuclear oxovanadium complex was synthesized, its composition and structure were confirmed by spectral studies and its efficacy was studied in streptozotocin-induced diabetic rats over a period of 30 days. The oral administration of the complex normalizes the blood glucose level in the diabetic rats and also maintains normoglycemia after a glucose load. The biochemical studies revealed that the complex is not toxic to the system. The nontoxic nature of this complex may be due to the presence of the vanadyl ions in an intact form. The study highlights the nontoxic and hypoglycemic effects of the new macrocyclic binuclear oxovanadium complex.     

A biomedically enriched collection of 7000 human ORF clones

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.