An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.     

Changing water use and adaptive strategies along rainfall gradients in Mediterranean lupins

• There is growing interest in harnessing the genetic and adaptive diversity of crop wild relatives to improve drought resilience of elite cultivars. Rainfall gradients exert strong selection pressure on both natural and agricultural ecosystems. Understanding plant responses to these facilitates crop improvement.
• Wild and domesticated narrow‐leafed lupin (NLL) collected along Mediterranean terminal drought stress gradients was evaluated under contrasting reproductive phase water supply in controlled field, glasshouse and cabinet studies. Plant phenology, growth and productivity, water use and stress responses were measured over time.
• There is an integrated suite of adaptive changes along rainfall gradients in NLL. Low rainfall ecotypes flower earlier, accumulate lower seed numbers, biomass and leaf area, and have larger root:shoot ratios than high rainfall ecotypes. Water‐use is lower and stress onset slower in low compared to high rainfall ecotypes. Water‐use rates and ecotypic differences in stress response (Ψleaf decline, leaf loss) are lower in NLL than yellow lupin (YL). To mitigate the effects of profligate water use, high rainfall YL ecotypes maintain higher leaf water content over declining leaf water potential than low rainfall ecotypes. There is no evidence for such specific adaptation in NLL.
• The data suggests that appropriate phenology is the key adaptive trait to rainfall gradients in NLL because of the flow‐on effects on biomass production, fitness, transpiration and stress onset, and the lack of physiological adaptations as in YL. Accordingly, it is essential to match phenology with target environment in order to minimize risk and maximize yield potential.

     

Genetic instability of industrial strains of Penicillium chrysogenum

Summary It has shown that several characteristics of high-producing industrial strains of Penicillium chrysogenum tend to segregate in the course of cultivation (slant-to-slant transfer). Segregation includes a decrease in the yield of penicillin, mean conidial size, mean size of the nuclei, and an increase in the proportion of morphologically wild-type colonies. These lower-producing segregants also have a higher sensitivity against ultraviolet radiation and, as shown by cytofluorometric methods, a lower DNA content in the condia, a decrease in phosphate uptkae and in the activity of extracellular alkaline phosphatases compared to high-producing strains. Obviously, during mutagenesis/selection programmes ploidy mutants have been selected, which entails an increase in the number of genes coding enzymes responsible for penicillin biosynthesis. In the absence of selection pressure these high-producing strains segregate to lower-producing strains by chromosome losses in the course of slant-to-slant transfers. Offprint requests to: W. Künkel     

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Extracellular Enzymes and Nutritional Physiology of Ampelomyces quisqualis Ces., Hyperparasite of Powdery Mildew, in vitro

In vitro, the powdery’ mildew hyperparasite Ampelomyces quisqualis produced constitutively two groups of extracellular enzymes; β-glucosidase, β-N-acetylglucosaminidase and acid phosphatase possessing unusual high molecular weights (M, about 340 000), and ribonuclease, β-l→3-glucanase and α-l→4-glucanase representing smaller molecules (M, about 15 000–55 000). As can be concluded from adsorption to Concanavalin A-Sepharose 4 B, these enzymes are of glycoprotein nature Furthermore, traces of phospholipase could be detected in all of the isolates tested, whereas only phosphorylated carbohydrate components and nucleotides including cAMP as nutrients. The results of these studies suggest that in the biotrophic phase of parasitization A. qwsqualis interferes with energy metabolism, protein synthesis, cell wall synthesis and, possibly, regulation of the host, whereas proteins and membranes remain nearly unaffected. Thus, depletion of the energy reserves of the powdery mildew may be mainly responsible for the degeneration of host ultrastructure which represents the beginning of the necrotrophic phase of parasitization.