Material and mechanical properties of bones deficient for fibrillin-1 or fibrillin-2 microfibrils

The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of fibrillin-1 or fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in fibrillin-1 or fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that fibrillin-1 plays a predominantly greater role than fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by fibrillin proteins. Our study therefore advances our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in fibrillin-1 or -2.     

Expanding hybrid zone between Solea aegyptiaca and Solea senegalensis: genetic evidence over two decades

Several marine hybrid zones have been described and studied during the last years. Assessing the movements of extending hybrid zones is central to improve our understanding of evolutionary processes. We have re-examined the hybrid zone between Solea aegyptiaca and Solea senegalensis that was first described 22 years ago in northern Tunisia when introgressed S. senegalensis individuals were found in the Gulf of Tunis, whereas locally caught S. aegyptiaca were genetically pure. Six population samples harvested both inside and outside the area where the two fish species coexist were genotyped for allozymes and exon-primed intron length polymorphism. Both types of markers were congruent and revealed that introgression takes place indeed in both directions. A high introgression level (36.4%) in the Bizerta lagoon and much less outside indicate that this is the main area where hybridization occurs while introgression clines towards the south in S. aegyptiaca and towards the north in S. senegalensis plead in favour of the existence of a unimodal hybrid zone. The higher introgression level calculated in the current study (when compared to 16% reported formerly) and the newly found introgressed S. aegyptiaca in Bizerta lagoon seem to indicate that the genetic exchanges occurring between the two taxa are evolving and not stabilized yet.     

Natural hybridization between extremely divergent chromosomal races of the common shrew (Sorex araneus, Soricidae, Soricomorpha): hybrid zone in Siberia

Chromosomal races of the common shrew differ in sets of metacentric chromosomes and on contact may produce hybrids with extraordinarily complex configurations at meiosis I that are associated with reduced fertility. There is an expectation that these may be some of the most extreme tension zones available for study and therefore are of interest as potential sites for reproductive isolation. Here, we analyse one of these zones, between the Novosibirsk race (characterized by metacentrics go, hn, ik, jl, mp and qr) and the Tomsk race (metacentrics gk, hi, jl and mn and acrocentrics o, p, q and r), which form hybrids with a chain-of-nine (CIX) and a chain-of-three (CIII) configuration at meiosis I. At the Novosibirsk-Tomsk hybrid zone, the CIX chromosomes form clines of 8.53 km standardized width on average, whereas the cline for the CIII chromosomes was 52.83 km wide. The difference in these cline widths fits with the difference in meiotic errors expected with the CIX and CIII configuration, and we produce estimates of selection against hybrids with these types of configurations, which we relate to dispersal and age of the hybrid zone. The hybrid zone is located at the isocline at 200 m altitude above sea level; this relationship between the races and altitude is suggested at both coarse and fine scales. This indicates adaptive differences between the races that may in turn have been promoted by the chromosome differences. Thus, the extreme chromosomal divergence between the Novosibirsk and Tomsk may be associated with genic differentiation, but it is still striking that, despite the large chromosomal differences, reproductive isolation between the Novosibirsk and Tomsk races has not occurred.     

周期性机械牵张对肺泡Ⅱ型上皮细胞Cyr61表达的影响

目的研究周期性牵张肺泡Ⅱ型上皮细胞株A549细胞对Cyr61表达的影响。方法对肺泡Ⅱ型上皮细胞株A549细胞施加周期性机械牵张应力。加载频率0.5 Hz,加载时间2 h,加载应力分别为5%,15%,30%。加载应力为15%,加载频率0.5 Hz,加载时间分别0,15 min,30 min,60 min,120 min。每个实验均设立空白对照即不给予机械应力。用PCR法测定Cyr61 mRNA的表达,用western法测定Cyr61蛋白含量。结果随着加载应力的增加和加载时间的延长,Cyr61蛋白含量和mRNA表达均增加(P<0.05);施加不同加载幅度后,Cyr61蛋白含量和mRNA表达均增加(P<0.05)。结论肺泡Ⅱ型上皮细胞IL-8的产生和释放与周期性的机械牵张应力呈强度和时间依赖性。     

Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

Fusicoccin affects cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

Epidermal peels isolated from sunflower hypocotyls provide a convenient model to study the relationship between cortical microtubule orientation and strain rate. Extension of peels can be modulated using chemical treatment and mechanical stress, i.e., by adding a chemical to the incubation medium and applying a load exceeding the yield threshold for irreversible (plastic) strain. In this study, peels were pre-incubated for ca. 12 h (long-term pre-incubation) or for 1 h (short-term pre-incubation). In the long-term pre-incubated peels, fusicoccin applied to the medium neither enhanced the rate of longitudinal plastic strain of loaded peels, nor affected microtubule orientation. However, fusicoccin increased the strain rate of short-term, pre-incubated peels and affected microtubule orientation in both extending (loaded) and non-extending (unloaded) peels. Without fusicoccin, microtubule orientation was generally longitudinal or steep, whereas in fusicoccin-treated unloaded peels it was transverse and oblique microtubules in peel portions corresponding to the apical part of the hypocotyl. Although the frequency of transverse orientation was increased through loading, there was no strong correlation between the rate of fusicoccin-induced strain and microtubule orientation. It is hypothesized that the insensitivity of long-term pre-incubated peels to fusicoccin with respect to strain rate is due to a lack of active plasma membrane H(+) -ATPases. Thus, the sensitivity of short-term, pre-incubated, unloaded (non-extending) peels to fusicoccin, with respect to microtubule orientation, indicates that orientation might be affected by electric currents resulting from fusicoccin stimulation of H(+) -ATPases.     

Serotonin modulates muscle function in the medicinal leech Hirudo verbana

The body wall muscles of sanguivorous leeches power mechanically diverse behaviours: suction feeding, crawling and swimming. These require longitudinal muscle to exert force over an extremely large length range, from 145 to 46 per cent of the mean segmental swimming length. Previous data, however, suggest that leech body wall muscle has limited capacity for force production when elongated. Serotonin (5-HT) alters the passive properties of the body wall and stimulates feeding. We hypothesized that 5-HT may also have a role in allowing force production in elongated muscle by changing the shape of the length-tension relationship (LTR). LTRs were measured from longitudinal muscle strips in vitro in physiological saline with and without the presence of 10 μM 5-HT. The LTR was much broader than previously measured for leech muscle. Rather than shifting the LTR, 5-HT reduced passive muscle tonus and increased active stress at all lengths. In addition to modulating leech behaviour and passive mechanical properties, 5-HT probably enhances muscle force and work production during locomotion and feeding.     

A cohesion/tension model for the gating of aquaporins allows estimation of water channel pore volumes in Chara

The water permeability (hydraulic conductivity; Lp) of turgid, intact internodes of Chara corallina decreased exponentially as the concentration of osmolytes applied in the medium increased. Membranes were permeable to osmolytes and therefore they could be applied on both sides of the plasma membrane at concentrations of up to 2.0 m (5.0 MPa of osmotic pressure). Organic solutes of different molecular size (molecular weight, MW) and reflection coefficients (σ_s) were used [heavy water HDO, MW: 19, σ_s: 0.004; acetone, MW: 58, σ_s: 0.15; dimethyl formamide (DMF), MW: 73, σ_s: 0.76; ethylene glycol monomethyl ether (EGMME), MW: 76, σ_s: 0.59; diethylene glycol monomethyl ether (DEGMME), MW: 120, σ_s: 0.78 and triethylene glycol monoethyl ether (TEGMEE), MW: 178, σ_s: 0.80]. The larger the molecular size of the osmolyte, the more efficient it was in reducing cell Lp at a given concentration. The residual cell Lp decreased with increasing size of osmolytes. The findings are in agreement with a cohesion/tension model of the osmotic dehydration of water channels (aquaporins; AQPs), which predicts both reversible exponential dehydration curves and the dependence on the size of osmolytes which are more or less excluded from AQPs (Ye, Wiera & Steudle, Journal of Experimental Botany 55, 449–461, 2004). In the presence of big osmolytes, dehydration curves were best described by the sum of two exponentials (as predicted from the theory in the presence of two different types of AQPs with differing pore diameters and volumes). AQPs with big diameters could not be closed in the presence of osmolytes of small molecular size, even at very high concentrations. The cohesion/tension theory allowed pore volumes of AQPs to be evaluated, which was 2.3 ± 0.2 nm³ for the narrow pore and between 5.5 ± 0.8 and 6.1 ± 0.8 nm³ for the wider pores. The existence of different types of pores was also evident from differences in the residual Lp. Alternatively, pore volumes were estimated from ratios between osmotic (P_f) and diffusional (P_d) water flow, yielding the number of water molecules (N) in the pores. N-values ranged between 35 and 60, which referred to volumes of 0.51 and 0.88 nm³/pore. Values of pore volumes obtained by either method were bigger than those reported in the literature for other AQPs. Absolute values of pore volumes and differences obtained by the two methods are discussed in terms of an inclusion of mouth parts of AQPs during osmotic dehydration. It is concluded that the mouth part contributed to the absolute values of pore volumes depending on the size of osmolytes. However, this can not explain the finding of the existence of two different types or groups of AQPs in the plasma membrane of Chara.     

Growth dynamics of mechanically impeded lupin roots: does altered morphology induce hypoxia?

BACKGROUND AND AIMS: Root axes elongate slowly and swell radially under mechanical impedance. However, temporal and spatial changes to impeded root apices have only been described qualitatively. This paper aims (a) to quantify morphological changes to root apices and (b) assess whether these changes pre-dispose young root tissues to hypoxia. METHODS: Lupin (Lupinus angustifolius) seedlings were grown into coarse sand that was pressurized through a diaphragm to generate mechanical impedance on growing root axes. In situ observations yielded growth rates and root response to hypoxia. Roots were then removed to assess morphology, cell lengths and local growth velocities. Oxygen uptake into excised segments was measured. KEY RESULTS: An applied pressure of 15 kPa slowed root extension by 75% after 10-20 h while the same axes thickened by about 50%. The most terminal 2-3 mm of axes did not respond morphologically to impedance, in spite of the slower flux of cells out of this region. The basal boundary of root extension encroached to within 4 mm of the apex (cf. 10 mm in unimpeded roots), while radial swelling extended 10 mm behind the apex in impeded roots. Oxygen demand by segments of these short, thick, impeded roots was significantly different from segments of unimpeded roots when the zones of elongation in each treatment were compared. Specifically, impeded roots consumed O2 faster and O2 consumption was more likely to be O2-limited over a substantial proportion of the elongation zone, making these roots more susceptible to O2 deficit. Impeded roots used more O2 per unit growth (measured as either unit of elongation or unit of volumetric expansion) than unimpeded roots. Extension of impeded roots in situ was O2-limited at sub-atmospheric O2 levels (21% O2), while unimpeded roots were only limited below 11% O2. CONCLUSIONS: The shift in the zone of extension towards the apex in impeded roots coincided with greater vulnerability to hypoxia even after soil was removed. Roots still encased in impeded soil are likely to suffer from marked O2 deficits.     

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.