Manipulation of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid biosynthesis pathways in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>: elevated GDP-mannose pyrophosphorylase activity enhances <Emphasis Type="SmallCaps">l</Emphasis>-ascorbate levels in red fruit

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff–Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.     

Development of a panel of genome-wide ancestry informative markers to study admixture throughout the Americas

Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2 > 0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.     

Enzymatic Dehalogenation of Pentachlorophenol by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> of the Microbial Community from Tannery Effluent

Four different bacterial isolates obtained from a stable bacterial consortium were capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in the chemostat. The degradation of PCP by bacterial strain was preceded through an oxidative route as indicated by accumulation of tetrachloro-ρ-hydroquinone and dichlorohydroquinone as determined by high performance liquid chromatography (HPLC). Among the four isolates, Pseudomonas fluorescens exhibited maximum degradation capability and enzyme production. PCP-monooxygenase enzyme was extracted from culture extract and fractionated by DEAE-cellulose ion exchange chromatography. The molecular weight of the enzyme, purified from Pseudomonas fluorescens, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography was found to be 24,000 Da. Received: 22 July 2002 / Accepted: 23 September 2002     

Reptile responses to fire and the risk of post-disturbance sampling bias

Altered fire regimes are a driver of biodiversity decline. To plan effective management, we need to know how species are influenced by fire and to develop theory describing fire responses. Animal responses to fire are usually measured using methods that rely on animal activity, but animal activity may vary with time since fire, potentially biasing results. Using a novel approach for detecting bias in the pit-fall trap method, we found that leaf-litter dependent reptiles were more active up to 6 weeks after fire, giving a misleading impression of abundance. This effect was not discovered when modelling detectability with zero-inflated binomial models. Two species without detection bias showed early-successional responses to time since fire, consistent with a habitat-accommodation succession model. However, a habitat specialist did not have the predicted low abundance after fire due to increased post-fire movement and non-linear recovery of a key habitat component. Interactions between fire and other processes therefore must be better understood to predict reptile responses to changing fire-regimes. We conclude that there is substantial bias when trapping reptiles after fire, with species that are otherwise hard to detect appearing to be abundant. Studies that use a survey method based on animal activity such as bird calls or animal movements, likely face a similar risk of bias when comparing recently-disturbed with control sites.     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

Relationship Between β-Amyloid and Mitochondrial Dynamics

Mitochondria as dynamic organelles undergo morphological changes through the processes of fission and fusion which are major factors regulating their functions. A disruption in the balance of mitochondrial dynamics induces functional disorders in mitochondria such as failed energy production and the generation of reactive oxygen species, which are closely related to pathophysiological changes associated with Alzheimer’s disease (AD). Recent studies have demonstrated a relationship between abnormalities in mitochondrial dynamics and impaired mitochondrial function, clarifying the effects of morphofunctional aberrations which promote neuronal cell death in AD. Several possible signaling pathways have been suggested for a better understanding of the mechanism behind the key molecules regulating mitochondrial morphologies. However, the exact machinery involved in mitochondrial dynamics still has yet to be elucidated. This paper reviews the current knowledge on signaling mechanisms involved in mitochondrial dynamics and the significance of mitochondrial dynamics in controlling associated functions in neurodegenerative diseases, particularly in AD.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

Abnormal Pelage Color in an Isolated Population of <Emphasis Type="Italic">Alouatta guariba clamitans</Emphasis> Cabrera, 1940 in South Brazil

We located 4 brown howlers (1 adult male, 2 adult females, and 1 juvenile male) showing abnormally lighter pelage in 3 social groups comprising 5, 6, and 9 individuals in a 20 ha-forest fragment in the State of Rio Grande do Sul, Brazil. Two additional groups composed only of normally colored individuals also live in the fragment, which is isolated from nearby fragments by 267–1009 m. They were the only brown howlers with abnormal pelage color out of a total of 386 individuals belonging to 67 groups in 21 fragments in the 5876-ha study area. The isolation of the forest fragment, its high howler density (2.2 individuals⁄ha), and large group size (8.8 ± 2.4 individuals) may decrease the likelihood of successful immigration into the population, leading to an increased probability of inbreeding that may facilitate the expression of rare alleles.