Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.     

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Pectin: cell biology and prospects for functional analysis

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.     

Recognition of base mismatches in DNA by 5,6-chrysenequinone diimine complexes of rhodium(III): a proposed mechanism for preferential binding in destabilized regions of the double helix

5,6-chrysenequinone diimine (chrysi) complexes of rhodium(III) have been shown to be versatile and specific recognition agents for mismatched base pairs in DNA. The design of these compounds was based on the hypothesis that the sterically expansive chrysi ligand, which should be too wide to readily intercalate into B-DNA, would bind preferentially in the destabilized regions of the DNA helix near base mismatches. In this work, this recognition hypothesis is comprehensively explored. Comparison of the recognition patterns of the complex [Rh(bpy)(2)(chrysi)](3+) with a nonsterically demanding analogue, [Rh(bpy)(2)(phi)](3+) (phi = 9,10-phenanthrenequinone diimine), demonstrates that the chrysi ligand does indeed disfavor binding to B-DNA and generate mismatch selectivity. Examination of mismatch recognition by [Rh(bpy)(2)(chrysi)](3+) in both constant and variable sequence contexts using photocleavage assays indicates that the recognition of base mismatches is influenced by the amount that a mismatch thermodynamically destabilizes the DNA helix. Thermodynamic binding constants for the rhodium complex at a range of mismatch sites have been determined by quantitative photocleavage titration and yield values which vary from 1 x 10(6) to 20 x 10(6) M(-)(1). These mismatch-specific binding affinities correlate with independent measurements of thermodynamic destabilization, supporting the hypothesis that helix destabilization is a factor determining the binding affinity of the metal complex for the mismatched site. Although not the only factor involved in the binding of [Rh(bpy)(2)(chrysi)](3+) to mismatch sites, a model is proposed where helix destabilization acts as the "door" which permits access of the sterically demanding intercalator to the base stack.     

The effects of carp (Cyprinus carpio L.) on sediment export from a small urban impoundment

Significantly higher concentrations of suspended inorganic matter were observed in the outflows than the inflows of three impoundments along an urban stream in southern Ontario during baseflow conditions. This pattern was observed during two summers at two of the reservoirs. After carp (Cyprinus carpio L.) were eliminated (490 kg ha^–1) from the third reservoir, significantly less suspended sediment was found in the outflow. Burrowing benthic invertebrates (Chironomus sp. and Tubificidae) which dominated the benthic fauna were replaced by less tolerant, more lithophilic taxa after carp removal. Tubifids dominated the fauna again after carp recolonized the impoundment. These data suggest that carp removal can have a significant positive effect on water quality.