Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast

Cytokinesis requires the wholesale reorganization of the cytoskeleton and secretion to complete the division of one cell into two. In the budding yeast Saccharomyces cerevisiae, the IQGAP-related protein Iqg1 (Cyk1) promotes cytokinetic actin ring formation and is required for cytokinesis and viability [1-3]. As the actin ring is not essential for cytokinesis or viability, Iqg1 must act by another mechanism [4]. To uncover this mechanism, a screen for high-copy suppressors of the iqg1 lethal phenotype was performed. CYK3 suppressed the requirement for IQG1 in viability and cytokinesis without restoration of the actin ring, demonstrating that CYK3 promotes cytokinesis through an actomyosin-ring-independent pathway. CYK3 encodes a novel SH3-domain protein that was found in association with the actin ring and the mother-bud neck. cyk3 null cells had misshapen mother-bud necks and were deficient in cytokinesis. In the cyk3 null strain, actin rearrangements associated with cytokinesis appeared normal, suggesting that the phenotype reflects a defect in secretory targeting or septal synthesis. Deletion of either cyk3 or hof1 alone results in a mild cytokinetic phenotype [5-7], but deletion of both genes resulted in lethality and a complete cytokinetic block, suggesting overlapping function. Thus, Cyk3 appears to be important for cytokinesis and acts potentially downstream of Iqg1.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.     

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.     

Activity changes of calmodulin and Ca2+-ATPase during low-temperature-induced anthocyanin accumulation in Alternanthera bettzickiana

Effect of low temperature on anthocyanin accumulation in seedlings of Alternanthera bettzickiana and activity changes of calmodulin (CaM) and Ca²⁺-ATPase under low temperature were studied. Results indicate that the increase of anthocyanin content was obviously paralleled not only by the activity of CaM but also by the activity of Ca²⁺-ATPase. In addition, seedlings were pretreated with CaM antagonist [chlorpromazine (CPZ)] before low-temperature treatment in order to further investigate whether CaM plays a role in anthocyanin accumulation. CPZ pretreatment inhibited the activity of CaM and Ca²⁺-ATPase and caused a reduction in anthocyanin levels. Hence, it is concluded that CaM and Ca²⁺-ATPase were directly correlated with anthocyanin accumulation under low temperature, Ca² ± CaM may be involved in low-temperature signal transduction leading anthocyanin synthesis.     

Depletion of macro-nutrients from rhizosphere soil solution by juvenile corn,cottonwood, and switchgrass plants

In situ sampling of rhizosphere solution chemistry is an important step in improving our understanding of soil solution nutrient dynamics. Improved understanding will enhance our ability to model nutrient dynamics and on a broader scale, to develop effective buffers to minimize nutrient movement to surface waters. However, only limited attention has been focused on the spatial heterogeneity and temporal dynamics of rhizosphere solution, and still less is known about how rhizosphere solution chemistry varies among plant species. Nutrients in rhizosphere soil solution and changes in root morphology of juvenile corn (Zea mays L. cv. Stine 2250), cottonwood (Populus deltoids L.), and switchgrass (Panicum virgatum L.) were monitored using mini-rhizotron technology. Plants were grown for 10 days in a fine-silty, mixed, superactive, mesic Cumulic Hapludoll (Kennebec series). Micro-samples (100–200 μL) of rhizosphere and bulk soil solution were collected at 24-h intervals at a tension of −100 kPa and analyzed for P, K, Ca, and Mg concentration using Capillary Electrophoresis techniques. Plants were harvested at the end of the 10-day period, and tissue digests analyzed for nutrient content by Inductively Coupled Plasma Spectroscopy. Corn plants produced roots that were 1.3 times longer than those of cottonwood, and 11.7 times longer than those of switchgrass. Similar trends were observed in number of root tips and root surface area. At the end of 10 days, rhizosphere solution P and K concentrations in the immediate vicinity of the roots (<1 mm) decreased by approximating 24 and 8% for corn, and 15 and 5% for cottonwood. A rhizosphere effect was not found for switchgrass. After correction for initial plant nutrient content, corn shoot P, K, and Mg were respectively 385, 132, and 163% higher than cottonwood and 66, 37, and 10% higher than switchgrass. Cottonwood shoot Ca concentration, however, was 68 to 133% higher than that of corn or switchgrass. There was no difference in root P concentration among the three species. Nutrient accumulation efficiency (μg nutrient mm⁻¹ root length) of cottonwood was 26 to 242% higher for P, 25 to 325% higher for Ca, and 41 to 253% higher for Mg than those of corn and switchgrass. However, K accumulation efficiency of corn was four to five times higher than that of the cottonwood and switchgrass. Nutrient utilization efficiency (mg of dry weight produced per mg nutrient uptake) of P, K, and Mg was higher in cottonwood than in corn and switchgrass. These differences are element-specific and depend on root production and morphology as well as plant nutrient status. From a practical perspective, the results of this study indicate that potentially significant differences in rhizosphere solution chemistry can develop quickly. Results also indicate that cottonwood would be an effective species to slow the loss of nutrients in buffer settings. An erratum to this article can be found at     

Cloning and biological activity of an anti-tumor peptide of Tumstatin

To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203 amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction, the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC). The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide has the potential to become a novel, potent anti-tumor agent. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报]