In vitro production of metabolism-enhancing phytoecdysteroids from <Emphasis Type="Italic">Ajuga turkestanica</Emphasis>

In order to develop a sustainable source of metabolism-enhancing phytoecdysteroids, cell suspension and hairy root cultures were established from shoot cultures of wild-harvested Ajuga turkestanica, a medicinal plant indigenous to Uzbekistan. Precursors of phytoecdysteroids (acetate, mevalonic acid cholesterol) or methyl jasmonate (an elicitor) were added to subculture media to increase phytoecdysteroid accumulation. In cell suspension cultures, 20-hydroxyecdysone (20E) content increased 3- or 2-fold with the addition of 125 or 250 μM methyl jasmonate, respectively, compared to unelicited cultures. Precursor addition, however, did not provoke phytoecdysteroid accumulation. In hairy root cultures, addition of sodium acetate, mevalonic acid, and methyl jasmonate, but not cholesterol, increased phytoecdysteroid content compared to unelicited cultures. Hairy root cultures treated with 150 mg l⁻¹ sodium acetate, or 15 or 150 mg l⁻¹ mevalonic acid, increased 20E content approximately 2-fold to 19.9, 20.4 or 21.7 μg mg⁻¹, respectively, compared to control (10.5 μg mg⁻¹). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 μg mg⁻¹), turkesterone (0.9 μg mg⁻¹) and cyasterone (8.1 μg mg⁻¹) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 μg ml⁻¹ hairy root extract increased protein synthesis by 25.7% or 31.1%, respectively, in a C2C12 mouse skeletal cell line. These results suggest that sustainable production of metabolically active phytoecdysteroid can be achieved through hairy root culture systems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

A novel statistical analysis of cnidocysts in acontiarian sea anemones (Cnidaria, Actiniaria) using generalized linear models with gamma errors

Comparative studies on cnidocysts, involving adequate statistical treatment, are very scarce. Classical statistical tests are frequently used assuming normal frequency distributions of capsule lengths, but many distributions are non-normal in acontiarian sea anemones. A traditional choice in these situations are non-parametric tests, although they are not as powerful as parametric tests. An extension of classical methods was developed by some authors; these models, called Generalized Linear Models (GLM), can be used under certain conditions with non-normal data. In view of the properties of our data, that are positive, skewed and with constant coefficient of variation, a GLM with gamma distribution and inverse link function was chosen to analyse the cnidae of acontia from the species Haliplanella lineata, Tricnidactis errans and Anthothoe chilensis. Graphical analysis of residuals showed that these assumptions were reasonable. This method allowed us to avoid transformation of data set and controversial cases in the limit of significance level. For this task, appropriate subroutines in GLIM language were written. In all cases highly significant differences were found between the specimens considered for every species and nematocyst type (b-rhabdoids, p-rhabdoids B1b and p-rhabdoids B2a).     

Activation of PKCbeta(II) and PKCtheta is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKCβ_II and PKCθ from cytosol to plasma membrane, and inhibition of PKCβ_II and PKCθ decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKCβ_II and PKCθ, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKCβ_II and PKCθ. Inhibition of PKCβ_II or PKCθ, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKCθ in VSMC proliferation is unique.     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.     

A compilation of soybean ESTs: generation and analysis.

Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.     

Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes

A critical stage in the optimization of in vitro maturation (IVM) is the selection of good quality oocytes. There exists a relationship between the size of the cumulus investment and the in vitro developmental ability of the cumulus–oocyte complex (COC), which provides a basis for the selection of the COCs. This study was designed to evaluate the effect of the number of cumulus cell layers which enclose the oocytes, on the in vitro maturation, cytoplasm quality and cumulus expansion of the ovine oocytes. Ovaries were obtained from an abattoir and transported to the laboratory within 1–2 h, at 37 °C. Oocytes (n = 535) were recovered by means of an aspiration pump (set at a flow rate of 10 mL H₂O/min), with a disposable 20 G needle attached. Oocytes were divided into four classes (classes I to IV – with more than 5, 3–4, 1–2 and no cumulus cell layers, respectively) and separately cultured in a TCM199 medium for 24 h. The morphology of oocytes was evaluated following in vitro culture (IVC) to assess cumulus expansion, cytoplasm quality (score I with a homogenous cytoplasm and II with granulated cytoplasm) and nuclear maturation stage. The percentage of maximum cumulus expansion for classes I to III oocytes were 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% respectively. The rate of meiotic resumption of oocytes in classes I to IV were 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% respectively. The proportion of oocytes with a cytoplasm quality I in oocyte classes I to IV were 62.8 ± 1.5, 59.4 ± 1.2, 36.4 ± 2.1 and 0.5 ± 1.1%, respectively. Results showed that the presence of ≥3 cumulus cell layers in the COC prior to IVM led to a better (p < 0.05) cumulus expansion, meiotic resumption and cytoplasmic maturation rate. Thus the morphological grading of immature ovine oocytes may be an appropriate selection criterion regarding their developmental ability.