Variation of extractives content in heartwood and sapwood of Eucalyptus globulus trees

The variation in extractives content in sapwood and heartwood was investigated among 12 trees in each of four commercial plantations of Eucalyptus globulus in central Portugal. The study was carried out at the 15% height level and extractions used successively dichloromethane, ethanol and water. At all sites, heartwood had significantly more extractives than sapwood, on average 3.8 and 2.4%, respectively. Most extractives consisted of ethanol soluble material (on average 52% of total extractives). Among the sites, there was a statistically significant difference in the content of extractives but the most important source of variation was the within-tree variation between sapwood and heartwood. Differences in the content of extractives were also observed among trees. A strong relation between extractives content and heartwood proportion was found. The potential loss of pulp yield and problems associated with accumulation of extractives are directly related to the heartwood proportion in the eucalypt stems. Forest management should take into account heartwood development and selection for minimising heartwood extractives.     

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10 mg/kg live weight and ketoconazole at a dosage of 10 mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ + inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.     

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15 mg/kg), or nine times the recommended dose rate (45 mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)(200) SNP and a decrease in the (TAC)(167) SNP. All worms genotyped were GAA at codon 198.     

Histochemical and Morphometric Analyses of the Musculature of the Forelimb of the Subterranean Rodent Ctenomys talarum (Octodontoidea)

Histochemical and morphometric analyses were performed to characterize the fibre‐type composition of two forelimb muscles of the South American subterranean rodent Ctenomys talarum. The studied muscles were the triceps lateralis, an extensor of the elbow, and the teres major, a flexor of the shoulder. It was found that these muscles had an elevated proportion of fast oxidative‐glycolytic (FOG) fibres, and lower proportions of slow oxidative (SO) and fast glycolytic (FG) fibres. This composition probably qualifies the teres major and triceps muscles to perform the sustained effort required in tunnelling excavation. The results were discussed considering published data on fibre‐type composition of mammals having different modes of life and digging behaviour. We here suggest that C. talarum has the potential of generating forces linked to rapid, powerful movements during sustained periods by means of an elevated proportion of FOG fibres, together with osteological changes that result in a great mechanical advantage of the forelimb muscles.     

Assessing heavy metal contamination in soils of the Zagreb region (Northwest Croatia) using multivariate geostatistics

The assessment of soil contamination and location of pollution sources represent a crucial issue in soil remediation. Topsoil samples were collected in the Zagreb area (Northwest Croatia) and the total contents of trace and major elements were determined. A multivariate geostatistical analysis was used to estimate soil chemical composition variability. Factorial Kriging Analysis (FKA) was used to investigate the scale-dependent correlation structure of some variables by modelling co-regionalization of ten chemical variables, co-kriging specific factors and mapping them. The FKA provided two regionalized factors at different spatial scales of variability: the first factor at shorter range for Zn, Pb, Cd, Cu and Ni indicated different sources of anthropogenic contamination, whereas Ca (mainly loading on the longer range factor) was related to the lithology and parent material composition. The methodology used has proved to be a useful tool to separate geological and anthropogenic causes of variation in soil heavy metal content and to identify common pollution sources.     

Genetic variation for waxy proteins and amylose content in Spanish spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.)

Spelt wheat is a neglected crop that could be used in the quality breeding of modern common wheat. One important aspect of this quality is the starch composition which is related to the waxy proteins. A collection of 420 accessions of Spanish spelt wheat was analysed for waxy protein composition by SDS–PAGE. Polymorphism was found in the three waxy proteins, detecting differences both in size and in activity, and a new waxy allele (Wx-D1g) was identified. Seed amylose content was also determined and significant differences were detected among the different allelic combinations. In general, the accessions carrying one or two waxy null alleles showed less amylose content. The variation found could be used to enlarge the genetic pool of common wheat, or to develop lines of spelt with different levels of amylose content.     

Cellular structure and chemical composition of cork from the Chinese cork oak (Quercus variabilis)

Quercus variabilis Blume, the Chinese cork oak, is an oak species with a thick cork outer bark. The cork is exploited at a limited scale in China and considered of lower quality than the commercial cork from Quercus suber. We studied an industrial cork granulate feedstock of Q. variabilis in relation to cellular structure and chemical composition and compared it to Q. suber cork under a material’s perspective. The cork of Q.variabilis has 1.1 % ash, 9.6 % extractives, 34.8 % suberin and 19.1 % lignin. The monosaccharide composition with shows a predominance of hemicelluloses: glucose 42.8 % of total neutral sugars, xylose 27.5 %, arabinose 15.4 %, galactose 9.0 %, mannose 4.0 %, rhamnose 1.2 %. The FT-IR spectrum shows the indicative peaks of suberin. The composition is overall similar to that of Q. suber cork. Q. variabilis cork has the typical cellular characteristics of bark cork tissues with a regular and radially aligned structure of cells without intercellular voids. Solid volume fraction was estimated at approximately 16 %. Compared with Q. suber, the Q. variabilis cork cells are smaller, the cell wall undulation and the overall row alignment less homogeneous, the cell surface is irregular and the solid volume proportion higher. The characteristics of Q. variabilis cork support its use as a cellular material for sealing, insulation and energy absorption, but the overall quality is lower than that of Q. suber cork. The negative impact of the higher density and structural lower uniformity at tissue and cell level should be evaluated for processing and product performance.     

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.     

Oestrogen,Progesterone and Oxytocin Receptors and COX‐2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase‐2 (COX‐2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post‐GnRH. Blood samples were collected daily for progesterone and estradiol‐17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX‐2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol‐17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down‐regulated until its lowest expression was reached on day 10 post‐GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX‐2 was highest from day 8 to 12 post‐GnRH in the luminal epithelium, in relation to the time of maximal PGF_2α release. Both steroid receptors populations and COX‐2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8–10 post‐induction of ovulation and the concomitant increase of COX‐2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.