Genetic diversity and structure of the critically endangered tree Dimorphandra wilsonii and of the widespread in the Brazilian Cerrado Dimorphandra mollis: Implications for conservation

We performed a comparative analysis of the genetic diversity and structure of two congeneric tree species, one critically endangered, with only 21 known individuals in the wild, Dimorphandra wilsonii, and the other widely distributed Dimorphandra mollis. Eight populations of D. mollis and all known trees of D. wilsonii, from three areas, were screened for variability with ISSR markers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's index were considerably lower in D. wilsonii (P = 40.0%, h = 0.124 and I = 0.190), as compared to D. mollis (P = 70.4%, h = 0.190 and I = 0.297). Bayesian clustering showed that D. wilsonii individuals are clustered in three populations, which had high differentiation among them. Several measures for their conservation were suggested: protection of all extant populations, ex situ conservation of seeds, production of saplings in nurseries and foundation of new populations in reserve areas.     

Evaluation of three indices for biofilm accumulation on complete dentures

doi:10.1111/j.1741‐2358.2009.00285.x
Evaluation of three indices for biofilm accumulation on complete dentures Objectives: The objective of this study was to evaluate the accuracy and reproducibility of three complete denture biofilm indices (Prosthesis Hygiene Index; Jeganathan et al. Index; Budtz‐Jørgensen Index) by means of a computerised comparison method. Background: Clinical studies into denture hygiene have employed a large number of biofilm indices among their outcome variables. However, the knowledge about the validity of these indices is still scarce. Materials and methods: Sixty‐two complete denture wearers were selected. The internal surfaces of the upper complete dentures were stained (5% erythrosine) and photographed. The slides were projected on paper, and the biofilm indices were applied over the photos by means of a scoring method. For the computerised method, the areas (total and biofilm‐covered) were measured by dedicated software (Image Tool). In addition, to compare the results of the computerised method and Prosthetic Hygiene Index, a new scoring scale (including four and five graded) was introduced. For the Jeganathan et al. and Budtz‐Jørgensen indices, the original scales were used. Values for each index were compared with the computerised method by the Friedman test. Their reproducibility was measured by means of weighed κ. Significance for both tests was set at 0.05. Results: The indices tested provided similar mean measures but they tended to overestimate biofilm coverage when compared with the computerised method (p < 0.001). Agreement between the Prosthesis Hygiene Index and the computerised method was not significant, regardless of the scale used. Jeghanathan et al. Index showed weak agreement, and consistent results were found for Budtz‐Jorgensen Index (κ = 0.19 and 0.39 respectively). Conclusion: Assessment of accuracy for the biofilm indices showed instrument bias that was similar among the tested methods. Weak inter‐instrument reproducibility was found for the indices, except for the Budtz‐Jørgensen Index. This should be the method of choice for clinical studies when more sophisticated approaches are not possible.     

Evaluation of serum protein electrophoresis in greater rhea (<Emphasis Type="Italic">Rhea americana</Emphasis> Linnaeus, 1758)

Serum proteins from 47 healthy greater rheas (Rhea americana; male and female) were separated by electrophoresis in order to characterize normal reference ranges. Determination of total protein concentration was performed through biuret reaction. The mean value of total serum protein was 4.4 g/dL. Absolute concentrations of serum proteins were determined by agarose gel electrophoretic fractioning. Five fractions were analyzed: albumin, alpha, beta1, beta2, and gamma globulins, and the average values in grams per deciliter were 2.39, 0.32, 0.45, 0.30, and 0.79, respectively.     

A novel synthetic na?ve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.Key words: human antibody library, phage display, oncofetal fibronectin, vascular tumor targeting, scFv antibody fragments, chelating recombinant antibody (CRAb)     

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.     

Evaluation of experimental cleanser solution of Ricinus communis: effect on soft denture liner properties

doi: 10.1111/j.1741‐2358.2010.00438.x Evaluation of experimental cleanser solution of Ricinus communis: effect on soft denture liner properties Objective: This study evaluated colour stability, hardness and roughness of soft denture liners after immersion in various cleansers. Materials and methods: Thirty specimens (14 mm × 4 mm) of Elite Soft Relining (ES) and Mucopren Soft (MS) were randomly immersed in distilled water at 37°C, sodium hypochlorite 1%, and an experimental Ricinus communis solution (RC) for 7, 15 and 183 continuous days. Results: anova (p < 0.05) and Tukey’s test indicated that after T7 (μ =8.79 ± 7.36); T15 (μ = 4.23 ± 2.62) and T183 (μ = 8.78 ± 3.16), MS presented a higher increase in hardness than ES. After T7, MS underwent an increase in roughness (μ = 0.09 ± 0.80); ES underwent a decrease (μ = ?0.08 ± 0.16). RC caused the smallest variation in roughness. After T15, both materials presented an increase in roughness. After T183, ES (μ = ?0.30 ± 0.48) presented a higher roughness variation than MS (μ = ?0.07 ± 0.32). Hypochlorite caused an increase in roughness (μ = 0.02 ± 0.19). Conclusion: After all periods ES presented higher colour alteration than MS; highest colour alteration was caused by hypochlorite. Both materials were more stable after immersion in RC.     

Fine-scale spatial genetic structure of Dalbergia nigra (Fabaceae), a threatened and endemic tree of the Brazilian Atlantic Forest

The Atlantic Forest is one of the most diverse ecosystems in the world and considered a hotspot of biodiversity conservation. Dalbergia nigra (Fabaceae) is a tree endemic to the Brazilian Atlantic Forest, and has become threatened due to overexploitation of its valuable timber. In the present study, we analyzed the genetic diversity and fine-scale spatial genetic structure of D. nigra in an area of primary forest of a large reserve. All adult individuals (N = 112) were sampled in a 9.3 ha plot, and genotyped for microsatellite loci. Our results indicated high diversity with a mean of 8.6 alleles per locus, and expected heterozygosity equal to 0.74. The co-ancestry coefficients were significant for distances among trees up to 80 m. The Sp value was equal to 0.017 and indirect estimates of gene dispersal distances ranged from 89 to 144 m. No strong evidence of bottleneck or effects of human-disturbance was found. This study highlights that long-term efforts to protect a large area of Atlantic Forest have been effective towards maintaining the genetic diversity of D. nigra. The results of this study are important towards providing a guide for seed collection for ex-situ conservation and reforestation programmes of this threatened species.     

Phenotypic plasticity to light of two congeneric trees from contrasting habitats: Brazilian Atlantic Forest versus cerrado (savanna)

The Brazilian Atlantic Forest is a typically multi-layer tropical forest, while cerrado (savanna) is a patchy habitat with different physiognomy. Despite these differences, both habitats have high light heterogeneity. Functional traits of Dalbergia nigra and D. miscolobium from the Atlantic Forest and cerrado, respectively, were evaluated under shade (25% of full sunlight) and full sunlight in a nursery experiment. We hypothesised that both species should benefit from high phenotypic plasticity in relation to light. Plasticity was estimated using the relative distance phenotypic index (RDPI). D. miscolobium had lower shoot growth under both light conditions, suggesting it has low competitive capacity in the forest environment, which could explain its limited ability to expand over areas of Atlantic Forest. The studied species exhibited photoprotection strategies under high light and improved light capture under low light. Stomatal conductance, ETR(max) (maximum electron transport rate), PPFD(sat) (saturating photosynthetically active photon flux density), chlorophyll and carotenoid content had higher RDPI than stem morphological traits. Although both species showed considerable phenotypic plasticity, D. miscolobium had higher RDPI for eight of 11 evaluated traits. This high plasticity could be one of the factors that explain the occurrence of this species in a wide range of environmental conditions, from open grassland to dense woodlands, and it could also reflect its adaptation to high light. D. nigra also had considerable plasticity and good growth performance in both shade and full sunlight, but its absence in areas of cerrado suggests that factors other than light limit its occurrence in these habitats.     

Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado

Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process.