EST analysis of gene expression in the tentacle of Cyanea capillata

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.     

Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.     

3-Pyrroline containing arylacetamides: a novel series of remarkably selective kappa-agonists

A series of 2-(substituted phenyl)-N-methyl-N-[(1S)-1-(substituted alkyl)-2-(1-(3-pyrrolinyl))ethyl]acetamides were synthesized and evaluated as highly selective kappa-agonists with K(i) values in low nanomolar range. 3-Pyrroline incorporated into the basic amino functionality in combination with 2-(methylthio)ethyl substituent on the carbon adjacent to the amide nitrogen remarkably enhanced the kappa-selectivity. 3,4-Dichlorophenyl derivative 1e was found the most potent and selective analgesic in this series with ED(50) value of 0.023 mg/kg.     

Separation methods for taurine analysis in biological samples

Taurine plays an important role in a variety of physiological functions, pharmacological actions and pathological conditions. Many methods for taurine analysis, therefore, have been reported to monitor its levels in biological samples. This review discusses the following techniques: sample preparation; separation and determination methods including high-performance liquid chromatography, gas chromatography, ion chromatography, capillary electrophoresis and hyphenation procedures. It covers articles published between 1990 and 2001.     

Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes

NPR1 is an essential regulator of plant systemic acquired resistance (SAR), which confers immunity to a broad-spectrum of pathogens. SAR induction results in accumulation of the signal molecule salicylic acid (SA), which induces defense gene expression via activation of NPR1. We found that in an uninduced state, NPR1 is present as an oligomer formed through intermolecular disulfide bonds. Upon SAR induction, a biphasic change in cellular reduction potential occurs, resulting in reduction of NPR1 to a monomeric form. Monomeric NPR1 accumulates in the nucleus and activates gene expression. Inhibition of NPR1 reduction prevents defense gene expression, whereas mutation of Cys82 or Cys216 in NPR1 leads to constitutive monomerization, nuclear localization of the mutant proteins, and defense gene expression. These data provide a missing link between accumulation of SA and activation of NPR1 in the SAR signaling pathway.     

Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ~21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.     

Antitumor promoting activities of 3-O-acyl-(-)epigallocatechins

As an exploratory investigation of antitumor promoting compounds, 3-O-acyl-(-)-epigallocatechins possessing a straight-, branched-, phenyl-inserted- or 1,4-phenylene-inserted-acyl chain of varying length from C4 to C18 were synthesized and evaluated their inhibitory effects against the activation of the Epstein-Barr virus early antigen (EBV-EA). It was indicated that the epigallocatechin derivatives having the straight- or branched-acyl chain of C8 to C11 carbon atoms achieve marked effects.     

Computational Design of the β-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization

Computational design has been used with mixed success for the design of protein surfaces, with directed evolution heretofore providing better practical solutions than explicit design. Directed evolution, however, requires a tractable high-throughput screen because the random nature of mutation does not enrich for desired traits. Here we demonstrate the successful design of the β-sheet surface of a red fluorescent protein (RFP), enabling control over its oligomerization. To isolate the problem of surface design, we created a hybrid RFP from DsRed and mCherry with a stabilized protein core that allows for monomerization without loss of fluorescence. We designed an explicit library for which 93 of 96 (97%) of the protein variants are soluble, stably fluorescent, and monomeric. RFPs are heavily used in biology, but are natively tetrameric, and creating RFP monomers has proven extremely difficult. We show that surface design and core engineering are separate problems in RFP development and that the next generation of RFP markers will depend on improved methods for core design.     

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.