Cerato-platanin protein is located in the cell walls of ascospores, conidia and hyphae of Ceratocystis fimbriata f. sp. platani

Cerato-platanin (CP), a protein of about 12.4 kDa from Ceratocystis fimbriata f. sp. platani (Cfp), accumulated in the mycelium and was located in the cell walls of Cfp ascospores, hyphae and conidia suggesting that this protein had a role in forming the fungal cell wall apart from the already known fact that it is secreted early in culture and elicits phytoalexin synthesis and/or plant cell death. The finding was obtained with three immunological techniques: a quantitative ELISA which determines the amount of CP in the mycelium, an immunofluorescence assay, and immunogold labelling to define the exact localization of CP in the Cfp cells.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

Cloning plants by seeds: Inheritance models and candidate genes to increase fundamental knowledge for engineering apomixis in sexual crops

Apomixis is desirable in agriculture as a reproductive strategy for cloning plants by seeds. Because embryos derive from the parthenogenic development of apomeiotic egg cells, apomixis excludes fertilization in addition to meiotic segregation and recombination, resulting in offspring that are exact replicas of the parent. Introgression of apomixis from wild relatives to crop species and transformation of sexual genotypes into apomictically reproducing ones are long-held goals of plant breeding. In fact, it is generally accepted that the introduction of apomixis into agronomically important crops will have revolutionary implications for agriculture. This review deals with the current genetic and molecular findings that have been collected from model species to elucidate the mechanisms of apomeiosis, parthenogenesis and apomixis as a whole. Our goal is to critically determine whether biotechnology can combine key genes known to control the expression of the processes miming the main components of apomixis in plants. Two natural apomicts, as the eudicot Hypericum perforatum L. (St. John's wort) and the monocot Paspalum spp. (crowngrass), and the sexual model species Arabidopsis thaliana are ideally suited for such investigations at the genomic and biotechnological levels. Some novel views and original concepts have been faced on this review, including (i) the parallel between Y-chromosome and apomixis-bearing chromosome (e.g., comparative genomic analyses revealed common features as repression of recombination events, accumulation of transposable elements and degeneration of genes) from the most primitive (Hypericum-type) to the most advanced (Paspalum-type) in evolutionary terms, and (ii) the link between apomixis and gene-specific silencing mechanisms (i.e., likely based on chromatin remodelling factors), with merging lines of evidence regarding the role of auxin in cell fate specification of embryo sac and egg cell development in Arabidopsis. The production of engineered plants exhibiting apomictic-like phenotypes is critically reviewed and discussed.     

Cytogenetic and molecular evaluation of centromere-associated DNA sequences from a marsupial (Macropodidae: Macropus rufogriseus) X chromosome

The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.     

Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol

The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose. However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product. In this study, the growth characteristics of F. oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations. Under aerobic conditions the maximum specific growth rate was found to be 0.043 h(-1), and the highest ethanol yield (1.66 mol/mol) was found under anaerobic conditions. During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions. The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden-Meyerhof-Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions. The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation. On the other hand, the presence of high levels of gamma-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions.     

Ghrelin regulates mitochondrial-lipid metabolism gene expression and tissue fat distribution in liver and skeletal muscle

Ghrelin is a gastric hormone increased during caloric restriction and fat depletion. A role of ghrelin in the regulation of lipid and energy metabolism is suggested by fat gain independent of changes in food intake during exogenous ghrelin administration in rodents. We investigated the potential effects of peripheral ghrelin administration (two times daily 200-micrograms [DOSAGE ERROR CORRECTED] sc injection for 4 days) on triglyceride content and mitochondrial and lipid metabolism gene expression in rat liver and muscles. Compared with vehicle, ghrelin increased body weight but not food intake and circulating insulin. In liver, ghrelin induced a lipogenic and glucogenic pattern of gene expression and increased triglyceride content while reducing activated (phosphorylated) stimulator of fatty acid oxidation, AMP-activated protein kinase (AMPK, all P < 0.05), with unchanged mitochondrial oxidative enzyme activities. In contrast, triglyceride content was reduced (P < 0.05) after ghrelin administration in mixed (gastrocnemius) and unchanged in oxidative (soleus) muscle. In mixed muscle, ghrelin increased (P < 0.05) mitochondrial oxidative enzyme activities independent of changes in expression of fat metabolism genes and phosphorylated AMPK. Expression of peroxisome proliferator-activated receptor-gamma, the activation of which reduces muscle fat content, was selectively increased in mixed muscle where it paralleled changes in oxidative capacities (P < 0.05). Thus ghrelin induces tissue-specific changes in mitochondrial and lipid metabolism gene expression and favors triglyceride deposition in liver over skeletal muscle. These novel effects of ghrelin in the regulation of lean tissue fat distribution and metabolism could contribute to metabolic adaptation to caloric restriction and loss of body fat.     

Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.