Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.     

Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.     

Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers

Two transgenic rice (Oryza sativa L.) lines, KMD1 and KMD2 at the R4 generation, transformed with a synthetic cry1Ab gene from Bacillus thuringiensis Berliner, were first evaluated for stem borer resistance in the field during the rice growing season of 1998 in two areas of Zhejiang Province, China. Both KMD1 and KMD2 were highly resistant to the stem borers Chilo suppressalis (Walker) and Scirpophaga incertulas (Walker), and were completely undamaged during the whole rice growing season. In contrast, damage to the plants of the untransformed parental control (Xiushui 11) was in the form of deadhearts or whiteheads. Under natural infestation by the C. suppressalis, the damage to control plants reached a peak of 88.7% of plants and 20.1% of tillers encountered with deadhearts. Under artificial and natural infestation of neonate striped stem borers at the vegetative stage and booting stage, 100% of plants and 25.6% of tillers, 78.9% of plants and 15.6% of productive tillers among artificially infested control plants were observed with the symptom of deadhearts and whiteheads, respectively. Damage to the control plants from artificial infestation by the S. incertulas reached a peak of 97.0% of plants and 22.9% of tillers damaged. The field research indicated that both KMD1 and KMD2 show great potential for protecting rice from attack by these two stem borers.     

Secoiridoid constituents from the fruits of Ligustrum lucidum

Two secoiridoid glucosides, lucidumosides A and B, as well as six known glucosides, oleoside dimethyl ester, ligustroside, oleuropein, nuezhenide, isonuezhenide, and neonuezhenide, were isolated from the fruits of Ligustrum lucidum. Their structures were elucidated by spectroscopic methods.     

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.     

Backbone dynamics of the human CC-chemokine eotaxin

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. ¹⁵N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. ¹⁵N longitudinal (R₁) and transverse (R₂) auto relaxation rates, heteronuclear ¹H-¹⁵N steady-state NOEs, and transverse cross-relaxation rates (_xy) were obtained at 30 °C for all resolved backbone secondary amide groups using ¹ H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (_m) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D/D) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1–19), the C-terminus (residues 68–73) and the loop connecting the first two -strands (residues 30–37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond–millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.