Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.     

Membrane enzyme systems responsible for the Ca(2+)-dependent phosphorylation of Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and the dephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase

H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca(2)(+) (K(0.5) = 0.9 microM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCalpha and PKCbetaII. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of alpha-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be approximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.     

The novel genetic disorder microhydranencephaly maps to chromosome 16p13.3-12.1

We studied a large consanguineous Anatolian family with children who exhibited hydranencephaly associated with microcephaly. The children were severely affected. This novel genetic disorder is autosomal recessive. We used autozygosity mapping to identify a locus at chromosome 16p13.3-12.1; it has a LOD score of 4.11. The gene locus is within a maximal 11-cM interval between markers D16S497 and D16S672 and within a minimal critical region of 8 cM between markers D16S748 and D16S490.     

Biological control agents for white grubs (Coleoptera: Scarabaeidae) in anticipation of the establishment of the Japanese beetle in California

We tested biological control agents for the control of 3rd-instar scarab turfgrass pests, both for the masked chafer Cyclocephala hirta LeConte and the Japanese beetle, Popillia japonica Newman. The former species is endemic in California whereas the latter, although not yet established, constitutes a permanent serious threat to agriculture and horticulture in California. We conducted experiments using C. hirta in California and P. japonica in New Jersey. A field trial conducted in 2 different California turfgrass sites compared the field persistence in the absence of hosts of Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, the milky disease bacterium, Paenibacillus (=Bacillus) popilliae (Dutky), and the entomopathogenic nematodes Steinernema kushidai Mamiya and Heterorhabditis bacteriophora Poinar to that of the organophosphate diazinon. Soil samples taken 0-70 d after applications were bio-assayed with P. japonica. Only diazinon and the entomopathogenic nematode S. kushidai caused substantial mortality and S. kushidai activity persisted significantly longer than diazinon activity. In greenhouse experiments, combinations of entomopathogenic nematode species usually resulted in additive mortality of scarab larvae. Combinations of S. kushidai and diazinon also resulted in additive mortality. In field trials, the efficacy of H. bacteriophora and especially S. kushidai and S. glaseri, was comparable to that of diazinon over 14-18 d. However, it is likely that at least S. kushidai would have outperformed diazinon over an extended period because of its longer persistence and potential for recycling in the hosts. S. kushidai, should it become commercially available, deserves further examination as an alternative to chemical white grub control especially as a highly compatible component of sustainable turfgrass management.     

Cu,Zn superoxide dismutase structure from a microbial pathogen establishes a class with a conserved dimer interface

Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged. To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide. We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A resolution. The structure reveals that the dimeric bacterial enzymes form a structurally homologous class defined by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key beta-barrel subunit fold with copper and zinc ions located at the base of a deep loop-enclosed active-site channel. Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs. Two metal-mediated crystal contacts yielded our C222(1) crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form. This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that specifically target periplasmic Cu,Zn SODs of bacterial pathogens.     

Influence of lignin and its degradation products on enzymatic hydrolysis of xylan

The influence of lignin, lignin model compounds, and black liquor from the kraft pulping process on the hydrolysis of xylan by xylanase was investigated. Addition of vanillic acid, acetovanillone, and protocatechuic acid increased the rate of hydrolysis of xylan by as much as 18–50% at low concentrations, but reached maxima at about 0.05% concentration. Addition of vanillin caused a 15% improvement in xylan hydrolysis, while addition of guaiacol more than doubled the hydrolysis rate. Increasing concentrations of either lignin or black liquor also increased the hydrolysis rate of xylan. Circular dichroism spectroscopy indicated a change in the structure of xylanase in the presence of black liquor.     

OCI-5/GPC3, a Glypican Encoded by a Gene That Is Mutated in the Simpson-Golabi-Behmel Overgrowth Syndrome, Induces Apoptosis in a Cell Line–specific Manner

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3–induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line– specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.     

The flowering-time geneFT and regulation of flowering inArabidopsis

Transition from vegetative to reproductive development (flowering) is one of the most important decisions during the post-embryonic development of flowering plants. More than twenty loci are known to regulate this process inArabidopsis. Some of these flowering-time genes may act at the shoot apical meristem to regulate its competence to respond to floral inductive signals and floral evocation. Genetic and phenotypic analyses of mutants suggest that the late-flowering geneFT may be a good candidate for such genes. To test this, we have cloned theFT gene using aFT-deficiency line associated with a T-DNA insertion. Cloned genes and loss-of-function mutants in hand, it is now possible to analyse the role ofFT and other genes in flowering at the biochemical and cellular levels as well as at the genetic level. The deduced FT protein has homology with TFL1 and CEN proteins believed to be involved in regulation of inflorescence meristem identity. Phylogenetic analysis suggests that theFT group and theTFL1/CEN group of genes diverged before the diversification of major angiosperm clades. This raises the interesting question of the evolutionary relationship between the regulation of vegetative/reproductive switching in the shoot apical meristem and the regulation of inflorescence architecture in angiosperms. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Fronitier of Plant Biology”     

Biodiversity maintenance mechanisms differ between native and novel exotic-dominated communities

In many systems, native communities are being replaced by novel exotic-dominated ones. We experimentally compared species diversity decline between nine-species grassland communities under field conditions to test whether diversity maintenance mechanisms differed between communities containing all exotic or all native species using a pool of 40 species. Aboveground biomass was greater in exotic than native plots, and this difference was larger in mixtures than in monocultures. Species diversity declined more in exotic than native communities and declines were explained by different mechanisms. In exotic communities, overyielding species had high biomass in monoculture and diversity declined linearly as this selection effect increased. In native communities, however, overyielding species had low biomass in monoculture and there was no relationship between the selection effect and diversity decline. This suggests that, for this system, yielding behaviour is fundamentally different between presumably co-evolved natives and coevolutionarily naive exotic species, and that native-exotic status is important to consider.