Transforming growth factor-beta1 enhanced vascular endothelial growth factor synthesis in mesenchymal stem cells

Angiogenesis is essential for transplantation of mesenchymal stem cells (MSCs). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors identified to date. Elevated VEGF levels in MSCs correlate with the potential of MSCs transplantation. As an indirect angiogenic agent, transforming growth factor-β1 (TGF-β1) plays a pivotal role in the regulation of vasculogenesis and angiogenesis. However, the effect of TGF-β1 on VEGF synthesis in MSCs is still unknown. Besides, the intracellular signaling mechanism by which TGF-β1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of MSCs to TGF-β1 stimulated the synthesis of VEGF. Meanwhile, TGF-β1 stimulated the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, Ly 294002, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt significantly attenuated the VEGF synthesis stimulated by TGF-β1. Additionally, U0126, a specific inhibitor of ERK1/2, also significantly attenuated the TGF-β1-stimulated VEGF synthesis. These results indicated that TGF-β1 enhanced VEGF synthesis in MSCs, and the Akt and ERK1/2 activation were involved in this process.     

Nerve growth factor promotes formation of lumen-like structures in vitro through inducing apoptosis in human umbilical vein endothelial cells

Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.     

The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies

Synthetic antibody libraries with restricted chemical diversity were used to explore the intrinsic contributions of four amino acids (Tyr, Ser, Gly and Arg) to the affinity and specificity of antigen recognition. There was no correlation between nonspecific binding and the content of Tyr, Ser or Gly in the antigen-binding site, and in fact, the most specific antibodies were those with the highest Tyr content. In contrast, Arg content was clearly correlated with increased nonspecific binding. We combined Tyr, Ser and Gly to generate highly specific synthetic antibodies with affinities in the subnanomolar range, showing that the high abundance of Tyr, Ser and Gly in natural antibody germ line sequences reflects the intrinsic capacity of these residues to work together to mediate antigen recognition. Despite being a major functional contributor to co-evolved protein-protein interfaces, we find that Arg does not contribute generally to the affinity of naïve antigen-binding sites and is detrimental to specificity. Again, this is consistent with studies of natural antibodies, which have shown that nonspecific, self-reactive antibodies are rich in Arg and other positively charged residues. Our findings suggest that the principles governing naïve molecular recognition differ from those governing co-evolved interactions. Analogous studies can be designed to explore the roles of the other amino acids in molecular recognition. Results of such studies should illuminate the basic principles underlying natural protein-protein interactions and should aid the design of synthetic binding proteins with functions beyond the scope of natural proteins.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.     

Age-related changes in muscle fiber type frequencies and cross-sectional areas in straightbred and crossbred rabbits

This study was designed to investigate the effects of the interaction among genetic group, sex and age on the frequencies and cross-sectional areas of myofiber types in rabbits. A total of 48 straightbred and crossbred Botucatu rabbits, males and females, were involved in a split plot design with a 2 × 2 (genetic groups × genders) factorial arrangement. Young rabbits were weaned at 35 days of age and sequentially slaughtered, four per genetic group × sex combination, at 42, 63 and 84 days of age. The flexor carpi radialis muscle was dissected, histological sections (10 μm) were obtained and the frequencies and cross-sectional areas of myofiber types: I, IIA and IIB/X were determined. An effect of the genetic group × sex × slaughter age interaction was found on the frequency distribution of myofiber types. A transition from type IIA to type IIB/X fibers was observed (P < 0.01) with advancing age, except in crossbred females, but the frequency of IIA fibers was already lower (57.3%) and of IIB/X fibers numerically higher (33.7%) in this group at 42 days. The proportions of IIA fibers in straightbred males, crossbred males and straightbred females decreased from 80.1%, 89.4% and 68.8% at 42 days to 43.9%, 52.3% and 40.1% at 63 days, respectively, whereas the proportions of type IIB/X fibers, in the same groups, increased from 10.3%, 1.6% and 22.3% at 42 days to 42.2%, 37.0% and 49.8% at 63 days, respectively. In all three age points, type IIA fibers showed the largest cross-sectional areas, followed by type I and IIB/X fibers. The cross-sectional areas of IIB/X fibers were larger in crossbreds, but no differences were found between genetic groups concerning fiber types IIA and I. All three types of fibers showed positive linear association with age, but relative to the initial area type IIB/X fibers presented a higher degree of hypertrophy (144% up to 84 days) than type IIA and I fibers (86% and 85%, respectively). The flexor carpi radialis muscle was, on average, heavier in crossbred than in straightbred females, but no difference was observed between crossbred and straightbred males. Differences in the weight of flexor carpi radialis muscle were attributed to the hypertrophy of type IIB/X fibers in the crossbreds.     

Reproductive biology of Clintonia udensis

To clarify the life history of Clintonia udensis, we investigated its reproductive systems and spatio‐temporal population structure. Pollination experiments and the observation of floral visitors revealed that C. udensis was compatible with both self‐ and outcross‐pollen, and it potentially produces seeds by insect‐mediated outcrossing in natural conditions. In addition, propagation by clonal reproduction from rhizomes was evident. In this study, it was clarified that C. udensis potentially propagates by sexual and asexual reproduction and maintains its population through a stable frequency of flowering. The differences in the dependence on each reproduction mode could be one of the contributing factors for creating a variety of population sizes and distribution patterns of ramets in populations.     

Growth of Salmonella on sprouting alfalfa seeds as affected by the inoculum size, native microbial load and Pseudomonas fluorescens 2-79

Aims: To investigate the growth of salmonellae on sprouting alfalfa seeds as affected by the inoculum size, microbial load and Pseudomonas fluorescens 2–79. Methods and Results: Alfalfa seeds pre‐inoculated with ≤10¹–10³ CFU g^?1 of salmonellae and with or without Ps. fluorescens 2–79 were sprouted in glass jars and the population of salmonellae were determined daily for up to 6 days. The population of salmonellae on germinating seeds reached the maximum 2–3 days after sprouting when total bacterial count reached the maximum (10⁹ CFU g^?1). The population of salmonellae on sprouting seeds not treated with Ps. fluorescens 2–79 showed a net increase of 3–4 log units. However, the population of salmonellae on alfalfa seeds treated with Ps. fluorescens 2–79 showed a net increase of only 1–2 log units. Disinfection of seeds with calcium hypochlorite enhanced the growth of salmonellae. Conclusions: Treatment of seeds with Ps. fluorescens 2–79 reduced the growth of salmonellae by 2–3 log units. Significance and Impact of the Study: The potential of Ps. fluorescens 2–79 as a biological agent for use in control of salmonellae on sprouting seeds was demonstrated and warrants further investigation.     

非洲菊盘状花雄蕊发育与舌状花生长着色的对应关系

非洲菊(Gerbern hybrida)头状花序由外轮舌状花和内轮盘状花构成。通过观察内轮盘状花雄蕊花粉囊和花粉粒的形态结构与发育顺序,和测定外轮舌状花花瓣的长度、宽度、花色素苷含量等,对它们之间的对应关系进行了研究。花序外轮舌状花花瓣开始着色时为P3期,此时第1轮盘状花出现成熟花粉粒。研究明确了内轮盘状花花粉粒发育与外轮舌状花生长时期和花色素苷积累的对应关系。     

Adaptive Growth of Gymnosperm Branches-Ultrastructural and Micromechanical Examinations

The top, the lateral and the underside of basal branch segments of two gymnosperm species, spruce (Picea abies [L.] Karst.) and yew (Taxus baccata L.), were studied with respect to possible adaptation in structural and mechanical properties. Microtensile tests were performed on thin wet foils, which were removed from the periphery of the branches. Structural parameters such as density and the microfibril angle in the S2-layer were examined to investigate the structure-function relationships of the branch wood. The top, the lateral and the underside of both branches showed significant differences in their structural and mechanical properties. However, no significant variations were observed as a function of age and size development. The findings were discussed in view of adaptive growth strategies of trees, including biomechanical constraints of a horizontally growing branch.