Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.     

A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes

A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.     

Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia)

Several experiments were conducted to investigate in vitro regeneration of adventious shoots from cultured leaves of Japanese pear (Pyrus pyrifolia). A protocol was developed and regeneration achieved from six cultivars. Leaves harvested from shoot cultures which had been preconditioned on B5 medium with 5 μM thidiazuron plus 0.25 μM gibberellic acid were placed on regeneration medium of the same composition. Frequency of regeneration per leaf was as high as 23% but cultivar and environmental factors influenced the result. More mature (basal) leaves regenerated more frequently than younger ones from the shoot tip. Leaf orientation during regeneration and photoperiod was not a strong influence but regeneration from leaf pieces was less than from uncut leaves. An alternative regeneration procedure was developed in which first, shoot cultures were grown on the preconditioning medium. Leaves of the intact shoot cultures were then induced to regenerate directly when adventitious shoots formed on leaves of the intact shoot culture leaves without excision. Adventitious shoots from both procedures developed into typical shoot cultures when transferred to shoot culture maintenance medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic utility of structural alterations found in the chloroplast genome of pear: hypervariable regions in a highly conserved genome

The genome structure of pear chloroplast DNA (cpDNA) is extremely highly conserved in comparison with that of other angiosperms, and therefore, relatively few phylogenetic analyses for pear (Pyrus spp.) have been carried out using cpDNA as a marker. In this study, we identified two hypervariable regions in intergenic spacers of cpDNA from 21 species in Pyrus. One of these regions is 857 bp in length and lies between the accD-psaI genes, and the other is a 904-bp region between the rps16-trnQ genes. The mutation rate of gaps for the two regions was 10 and 26 times higher, respectively, than the base change rate. Twenty-five haplotypes were revealed among 21 species in Pyrus by 36 mutations found in the two regions. These included 27 gaps and 9 base changes but excluded cpSSRs. Phylogenetic relationships between the 25 haplotypes were generated by haplotype network analysis. The 25 haplotypes represented three groups (types A–C) with two large deletions, one 228 bp in length between the accD-psaI genes and the other 141 bp between the rps16-trnQ genes. Types A and B consisted mostly of pears native to East and South Asia. Type C contained mainly Pyrus communis and wild relatives native to Europe, West and Central Asia, Russia, and Africa. Type B might have diverged from primitives such as pea pears in type A. Phylogenetic utility of structural alterations (gaps) occurring in the hypervariable regions of Pyrus cpDNA is discussed.     

Developmental changes in the regulation of calcium-dependent neurite outgrowth

Intracellular calcium ions (Ca²⁺) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni²⁺, Cd²⁺, or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP₃R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca²⁺-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP₃R-mediated pathway to a RyR-mediated pathway.     

Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori

Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori. Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B. licheniformis cell wall. The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity. The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine-N-acetylmuramic acid units with peptide side chains.     

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.     

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens

 The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed. Received: 5 April 1999 / Revision received: 13 September 1999 / Accepted: 6 October 1999     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Introgression between native and prehistorically naturalized (archaeophytic) wild pear (<Emphasis Type="Italic">Pyrus</Emphasis> spp.) populations in Northern Tohoku,Northeast Japan

Hybridization between native and cultivated species is a concern in conservation biology. However, detecting such hybridization and distinguishing true natives from prehistorically naturalized species based on phenotypic characteristics is difficult. Here, we report on introgression between native and prehistorically introduced pear (Pyrus) species in Northern Tohoku (northern end of Honshu Island), Japan. We analyzed 20 microsatellites in 226 wild, seemingly wild, or cultivated materials. Phenetic analysis showed that wild Japanese populations of P. ussuriensis var. ussuriensis in Northern Tohoku, previously considered true natives based on morphology and phytogeography, differed from those in continental Asia, confirming their nativeness. However, Bayesian inference of population structures showed that Japanese P. ussuriensis was genetically admixed with two genetic clusters: true native P. ussuriensis var. ussuriensis and prehistorically introduced P. pyrifolia. Even in the Kitakami Mountains, where true native populations of var. ussuriensis are believed to persist, most wild trees were at least somewhat admixed. Prehistorically introduced then naturalized plants are treated as natives in Japan’s conservation management, and some are considered endangered. However, introgression of prehistorically naturalized P. pyrifolia into threatened native P. ussuriensis var. ussuriensis has occurred. This paper examines the implications for conservation management.