Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense related genes. To understand the cellular mechanism controlling defense response better, a novel pathogenesis-related (PR) gene and putative cell wall protein gene, CaTin2, was isolated through differential screening of a hot pepper cDNA library and characterized. CaTin2 gene was locally and systemically induced in hot pepper plants upon TMV-P₀ inoculation which induces HR. However, CaTin2 gene wasn't regulated by bacterial HR-specific signal pathway. The full-length cDNA for CaTin2, which is 864 nucleotides long, contained the open reading frame of 200 amino acids including cell wall targeting sequences of 26 amino acids. CaTin2 gene has no sequence similarity with other cell wall protein genes except the signal sequence and exists as only one copy in hot pepper genome. CaTin2 gene contains repeated helix-turn-helix motif consisting of 39 amino acids. CaTin2 mRNA accumulation was induced in response to various treatments such as ethylene, SA, MeJA, ABA, methyl viologen, NaCl and wounding at early time points. Subcelluar localization of CaTin2 was confirmed in the cell wall in hot pepper leaves by making CaTin2::smGFP fusion protein. The transgenic plants overexpressing CaTin2 cDNA were resistant to TMV and CMV inoculation. From these results, CaTin2 gene may encode a virus-related new cell wall protein member.     

Molecular basis of the pharmacological difference between rat and human bombesin receptor subtype-3 (BRS-3)

We cloned the gene and cDNA for rat bombesin receptor subtype-3 (BRS-3) and characterized its mRNA expression pattern and pharmacological properties. Despite the high degree of sequence similarity (80% identical), rat and human BRS-3 differ markedly in their pharmacological properties. Although the natural ligand for BRS-3 is still unknown, a synthetic peptide, dY-Q-W-A-V-(beta-A)-H-F-Nle-amide (dY-bombesin), activates human BRS-3 with an EC(50) of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC(50) = 2 microM). To understand the molecular basis of this pharmacological difference, we constructed chimeric receptors in which individual extracellular loops of rat BRS-3 were replaced with the corresponding human sequences. Switching the N-terminal region or the second extracellular loop did not significantly change receptor properties. However, switching the third extracellular loop (E3) in the rat BRS-3 resulted in a chimeric receptor (RB3-E3) that behaved almost identically to human BRS-3. RB3-E3 bound dY-bombesin with high affinity (K(i) = 1.2 +/- 0.7 nM), and was activated by dY-bombesin with high potency (EC(50) = 1.8 +/- 0.5 nM). Within the E3 loop, mutation of Y(298)E(299)S(300) to S(298)Q(299)T(300) (RB3-SQT) or of D(306)V(307)P(308) to A(306)M(307)H(308) (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A(302)E(303) to V(302)D(303) or of V(310)V(311) to I(310)F(311) had little effect on the dY-bombesin potency. These results indicate that the sequence variation in the E3 loop is responsible for the species difference between rat and human BRS-3, and multiple residues in the E3 loop are involved in interactions with the agonist dY-bombesin.     

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [¹⁴C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [³H]glycerol, [³²P]Pi, [¹⁴C]myristic acid, and [¹⁴C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.     

DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca.

Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined. Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants. E1 and E4 were expressed 18- and 4-fold higher, respectively, in S. lividans than in E. coli. Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose. E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose. It readily cleaves cellotetraose to cellobiose. E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C. fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E. Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them. Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins. The catalytic domains of E1 and E4 are not similar to those of T. fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons. This sequence has been identified previously as the binding site for a protein that regulates induction.     

High sensitivity EndoV mutation scanning through real-time ligase proofreading

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5′ terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.     

Comparative Genetics of Functional Trinucleotide Tandem Repeats in Humans and Apes

Several human neurodegenerative disorders are caused by the expansion of polymorphic trinucleotide repeat regions. Many of these loci are functional short tandem repeats (STRs) located in brain-expressed genes, and their study is thus relevant from both a medical and an evolutionary point of view. The aims of our study are to infer the comparative pattern of variation and evolution of this set of loci in order to show species-specific features in this group of STRs and on their potential for expansion (therefore, an insight into evolutionary medicine) and to unravel whether any human-specific feature may be identified in brain-expressed genes involved in human disease. We analyzed the variability of the normal range of seven expanding STR CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and two nonexpanding polymorphic CAG loci (KCNN3 and NCOA3) in humans, chimpanzees, gorillas, and orangutans. The study showed a general conservation of the repetitive tract and of the polymorphism in the four species and high heterogeneity among loci distributions. Humans present slightly larger alleles than the rest of species but a more relevant difference appears in variability levels: Humans are the species with the largest variance, although only for the expanding loci, suggesting a relationship between variability levels and expansion potential. The sequence analysis shows high levels of sequence conservation among species, a lack of correspondence between interruption patterns and variability levels, and signs of conservative selective pressure for some of the STR loci. Only two loci (SCA1 and SCA8) show a human specific distribution, with larger alleles than the rest of species. This could account, at the same time, for a human-specific trait and a predisposition to disease through expansion.This article contains online supplementary material.     

中药王不留行化学成分的研究Ⅲ

从经产麦蓝菜（Ｖａｃｃａｒｉａ ｓｅｇｅｔａｌｉｓ（Ｎｅｃｋ）Ｇａｒｃｈｅ．）的种子王不留行中分离得到５个化合物,经波谱分析和化学方法分别鉴定为化学方法分别鉴定为：ＶａｃｃａｒｏｓｉｄｅＢ（１）、Ｃ（２）、Ｄ、（３）,６－Ｎ－ｍｅｔｈｙｌａｄｅｎｏｓｉｎｅ（４）和Ｎ,Ｎ－ｄｉｍｅｔｈｙｌ－Ｌ－ｔｒｙｐｔｏｐｈａｎ（５）。其中化合物４和５为首次从该植物中分得。     

A micromethod for the stereochemical analysis of fatty acid desaturase-mediated sulfoxidation reactions

The stereochemistry of fatty acid desaturase-mediated sulfoxidation can be evaluated at micromolar concentrations of analyte using (19)F NMR in combination with a chiral shift reagent: (S)-(+)-MPAA.     

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.     

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.