Suppressors of zyg-1 define regulators of centrosome duplication and nuclear association in Caenorhabditis elegans

In Caenorhabditis elegans, the kinase ZYG-1 is required for centrosome duplication. To identify factors that interact with ZYG-1, we used a classical genetic approach and identified 21 szy (suppressor of zyg-1) genes that when mutated restore partial viability to a zyg-1 mutant. None of the suppressors render animals completely independent of zyg-1 activity and analysis of a subset of the suppressors indicates that all restore the normal process of centrosome duplication to zyg-1 mutants. Thirteen of these suppressor mutations confer phenotypes of their own and cytological examination reveals that these genes function in a variety of cellular processes including cell cycle timing, microtubule organization, cytokinesis, chromosome segregation, and centrosome morphology. Interestingly, several of the szy genes play a role in attaching the centrosome to the nuclear envelope. We have found that one such szy gene is sun-1, a gene encoding a nuclear envelope component. We further show that the role of SUN-1 in centrosome duplication is distinct from its role in attachment. Our approach has thus identified numerous candidate regulators of centrosome duplication and uncovered an unanticipated regulatory mechanism involving factors that tether the centrosome to the nucleus.     

A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves

An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process.     

A monoclonal antibody and the lectin wheat germ agglutinin induce zoospore encystment in Pythium porphyrae, a marine microbial pathogen

Pythium porphyrae (Oomycota) is a microbial pathogen which causes red rot disease in the commercially cultivated red seaweed Porphyra. This disease is initiated by the motile zoospores of the fungus, which it has been suggested to recognize and process host specific signals by membrane bound receptors. Monoclonal antibodies (MAbs) were developed against the surface components of zoospores and cysts of this fungus in order to try and identify the putative receptor molecules involved in the zoospore encystment process. Screening of MAbs by immunofluorescence assays has revealed three different patterns of surface epitope binding, while labeling of zoospore and cysts components by FITC-conjugated lectins has identified different carbohydrate moieties. Of the MAbs and lectins tested, MAb 1A3 and wheat germ agglutinin have induced zoospore encystment under in vitro conditions. MAb 1A3 identified a 109 KDa band of a glycoprotein in western blot analysis which could be a putative receptor responsible for the induction of zoospore encystment.     

A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [³²P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.     

Spermatinamine, the first natural product inhibitor of isoprenylcysteine carboxyl methyltransferase, a new cancer target

Isoprenylcysteine methyltransferase (Icmt) catalyzes the carboxyl methylation of oncogenic proteins in the final step of a series of post-translational modifications. The inhibition of Icmt provides an attractive and novel anticancer target. A natural product high-throughput screening campaign was conducted to discover inhibitors of Icmt. The Australian marine sponge, Pseudoceratina sp., yielded spermatinamine, a novel alkaloid with a bromotyrosyl-spermine-bromotyrosyl sequence, as the bioactive constituent. Its structure was determined by 1D and 2D NMR spectroscopy. Spermatinamine is the first natural product inhibitor of Icmt.     

The interaction between two Arabidopsis polyadenylation factor subunits involves an evolutionarily-conserved motif and has implications for the assembly and function of the polyadenylation complex

The polyadenylation factor subunit "Factor Interacting with Poly(A) polymerase" (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.     

Factors Affecting the Ability of the Spectral Domain Optical Coherence Tomograph to Detect Photographic Retinal Nerve Fiber Layer Defects

Purpose

To evaluate the ability of normative database classification (color-coded maps) of spectral domain optical coherence tomograph (SDOCT) in detecting wedge shaped retinal nerve fiber layer (RNFL) defects identified on photographs and the factors affecting the ability of SDOCT in detecting these RNFL defects.

Methods

In a cross-sectional study, 238 eyes (476 RNFL quadrants) of 172 normal subjects and 85 eyes (103 RNFL quadrants with wedge shaped RNFL defects) of 66 glaucoma patients underwent RNFL imaging with SDOCT. Logistic regression models were used to evaluate the factors associated with false positive and false negative RNFL classifications of the color-coded maps of SDOCT.

Results

False positive classification at a p value of <5% was seen in 108 of 476 quadrants (22.8%). False negative classification at a p value of <5% was seen in 16 of 103 quadrants (15.5%). Of the 103 quadrants with RNFL defects, 64 showed a corresponding VF defect in the opposite hemisphere and 39 were preperimetric. Higher signal strength index (SSI) of the scan was less likely to have a false positive classification (odds ratio: 0.97, p = 0.01). Presence of an associated visual field defect (odds ratio: 0.17, p = 0.01) and inferior quadrant RNFL defects as compared to superior (odds ratio: 0.24, p = 0.04) were less likely to show false negative classifications.

Conclusions

Scans with lower signal strengths were more likely to show false positive RNFL classifications, and preperimetric and superior quadrant RNFL defects were more likely to show false negative classifications on color-coded maps of SDOCT.     

Cytotoxic agarofurans from the seeds of the Australian rainforest vine Celastrus subspicata

An anti-inflammatory active extract from the seeds of the Australian rainforest vine Celastrus subspicata was identified from high throughput screening of a plant and marine invertebrate extract library using an assay which detects down regulation of expression of pro-inflammatory genes associated with the glucocorticoid receptor ligand complex. The anti-inflammatory properties of the extract were found to be associated with a group of fatty acids that were present in the extract in high yield. Two agarofurans were also isolated and shown to possess minimal anti-inflammatory activity and to be cytotoxic towards HeLa cancer cells.     

(−)-Dibromophakellin: An α2B adrenoceptor agonist isolated from the Australian marine sponge,Acanthella costata

Bioassay-guided fractionation of the organic extract from the marine sponge Acanthella costata resulted in the isolation of the known natural product, (?)-dibromophakellin (1). Using a fluorescence imaging plate reader (FLIPR) based assay, compound 1 was identified as displaying agonist activity against the α_2B adrenoceptor, with an EC₅₀ of 4.2 μM. Debromination and Suzuki–Miyaura coupling reactions were undertaken in order to provide structure activity data about the pyrrole ring of this marine metabolite. These synthetic studies generated the known natural product analogues, (?)-phakellin (2), and (?)-monobromophakellin (3), along with the new synthetic derivatives (?)-4-bromo-5-phenylphakellin (5) and (?)-4,5-diphenylphakellin (6). Substitution of the C-5 Br of 1 with H (2 and 3) or phenyl (5 and 6) resulted in loss of activity indicating that Br at C-5 is required for agonist activity.