Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract -- critical parameters of protein isolation from anaerobic culture

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-β-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.     

The synthetic strigolactone GR24 influences the growth pattern of phytopathogenic fungi

Strigolactones that are released by plant roots to the rhizosphere are involved in both plant symbiosis with arbuscular mycorrhizal fungi and in plant infection by root parasitic plants. In this paper, we describe the response of various phytopathogenic fungi to the synthetic strigolactone GR24. When GR24 was embedded in the growth medium, it inhibited the growth of the root pathogens Fusarium oxysporum f. sp. melonis, Fusarium solani f. sp. mango, Sclerotinia sclerotiorum and Macrophomina phaseolina, and of the foliar pathogens Alternaria alternata, Colletotrichum acutatum and Botrytis cinerea. In the presence of this synthetic strigolactone, intense branching activity was exhibited by S. sclerotiorum, C. acutatum and F. oxysporum f. sp. melonis. Slightly increased hyphal branching was observed for A. alternata, F. solani f. sp. mango and B. cinerea, whereas suppression of hyphal branching by GR24 was observed in M. phaseolina. These results suggest that strigolactones not only affect mycorrhizal fungi and parasitic plants, but they also have a more general effect on phytopathogenic fungi.     

Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.     

Exploring phylogeography and species limits in the Altai vole (Rodentia: Cricetidae)

Natural hybridization between species is not a rare event. In arvicoline rodents, hybridization is known to occur in the wild and/or in captivity. In the Microtus arvalis group, cytogenetic studies revealed that there were two distinct chromosomal forms (2n = 46 but a different fundamental number of autosomes). These forms have been attributed to two cryptic species: the common (arvalis) and Altai (obscurus) voles. Recently, individuals with intermediate karyotypes (F₁ and backcrosses) were discovered in central European Russia, and, for this reason, other studies have regarded obscurus and arvalis as conspecific. In the present study, to address the question of the species limits in the Altai vole and to infer its evolutionary history, a phylogeographical analysis combined with multivariate morphometric methods and original chromosome data was performed. Two obscurus lineages were identified: the Sino‐Russian and South Caucasian lineages. Both lineages are characterized by low genetic diversity, resulting, in the former, from a past bottleneck event caused by encroaching periglacial areas and, in the latter, from recent rapid population divergence. Introgressive hybridization between the Altai and common voles appears to be the result of a secondary contact following the Last Glacial Maximum in central European Russia. Despite the fact that speciation is an ongoing process in most arvicoline species, the common and Altai voles are genetically divergent, morphologically and karyologically distinct, and exhibit contrasting evolutionary histories. For all these reasons, they should be ranked as species: M. arvalis and M. obscurus. © 2013 The Linnean Society of London     

Carbon sequestration and turnover in soil under the energy crop Miscanthus: repeated 13C natural abundance approach and literature synthesis

The stability and turnover of soil organic matter (SOM) are a very important but poorly understood part of carbon (C) cycling. Conversion of C₃ grassland to the C₄ energy crop Miscanthus provides an ideal opportunity to quantify medium‐term SOM dynamics without disturbance (e.g., plowing), due to the natural shift in the δ¹³C signature of soil C. For the first time, we used a repeated ¹³C natural abundance approach to measure C turnover in a loamy Gleyic Cambisol after 9 and 21 years of Miscanthus cultivation. This is the longest C₃–C₄ vegetation change study on C turnover in soil under energy crops. SOM stocks under Miscanthus and reference grassland were similar down to 1 m depth. However, both increased between 9 and 21 years from 105 to 140 mg C ha^?1 (P < 0.05), indicating nonsteady state of SOM. This calls for caution when estimating SOM turnover based on a single sampling. The mean residence time (MRT) of old C (>9 years) increased with depth from 19 years (0–10 cm) to 30–152 years (10–50 cm), and remained stable below 50 cm. From 41 literature observations, the average SOM increase after conversion from cropland or grassland to Miscanthus was 6.4 and 0.4 mg C ha^?1, respectively. The MRT of total C in topsoil under Miscanthus remained stable at ~60 years, independent of plantation age, corroborating the idea that C dynamics are dominated by recycling processes rather than by C stabilization. In conclusion, growing Miscanthus on C‐poor arable soils caused immediate C sequestration because of higher C input and decreased SOM decomposition. However, after replacing grasslands with Miscanthus, SOM stocks remained stable and the MRT of old C₃‐C increased strongly with depth.     

Variation of sperm morphology in Pacific oyster precludes its use as a species marker but enables intraspecific geo-authentification and aquatic monitoring

According to recent reports, shell morphology is unreliable for the identification of oysters because of the high phenotypic plasticity of these bivalves. Using COI DNA barcoding and sperm morphology, we reinvestigated the species validity of wild Pacific oyster Crassostrea gigas habituating the Peter the Great Bay (Sea of Japan). DNA barcoding confirmed the species validity of samples collected. Application of the single sperm pattern was not possible for species identification due to pronounced sperm plasticity being found. Six sperm morphs were discovered in the testes of each oyster collected. The amount of abundant sperm morphs and the type of the most dominant sperm pattern are particular to geographical localities that are individual depending on the environmental factors. Ecological monitoring of marine areas and commercially assigned intraspecific geo-authentification of the Pacific oyster seems possible based on the analysis of this species’ heterogenic sperm. Further work will be needed to test if sperm heterogeneity exists in other Ostreidae species and if heterogenic sperms could be used for interspecific analysis.