C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development

Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.     

Phosphorylation of STAT-3 in response to basic fibroblast growth factor occurs through a mechanism involving platelet-activating factor, JAK-2, and Src in human umbilical vein endothelial cells. Evidence for a dual kinase mechanism

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with multiple pathological and physiological effects. We have shown that basic fibroblast growth factor (bFGF) supplementation induces rapid proliferation of human umbilical vein endothelial cells (HUVEC), which is reduced upon removal of bFGF or by bFGF immunoneutralization. The PAF receptor antagonist LAU-8080 inhibited bFGF-stimulated HUVEC proliferation, indicating the involvement of PAF in the bFGF-mediated signaling of HUVEC. Although FGF receptor phosphorylation was not affected by LAU-8080, the bFGF-mediated prolonged phosphorylation, and activation of Erk-1 and -2 were attenuated. Phosphorylation of STAT-3 was observed in the presence of PAF or bFGF, which was attenuated by PAFR antagonists. PAF-induced STAT-3 phosphorylation observed in HUVEC pretreated with either Src inhibitor PP1 or JAK-2 inhibitor AG-490 indicated (i) immediate (1 min) phosphorylation of STAT-3 is dependent on Src, (ii) JAK-2-dependent STAT-3 phosphorylation occurs after the delayed (30 min) PAF exposure, and (iii) prolonged (60 min) STAT-3 phosphorylation may be either through Src and/or JAK-2. Attenuation of the STAT-3 phosphorylation by the PAFR antagonists indicated signaling through the PAF receptor. Taken together, these findings suggest the production of PAF is important for bFGF-mediated signaling and that a dual kinase mechanism is involved in the PAF-mediated signal transduction cascade.     

Ontogeny of enhancing factor in mouse intestines and skin.

Enhancing Factor (EF) is a 14 kDa protein isolated from mouse small intestines, which enhances the binding of 125I-EGF to A431 cells. This observation as well as our earlier in vitro studies have indicated that EF is a modulator of EGF. In adult mice, localization of EF by immunohistochemistry shows it is present predominantly in the Paneth cells of the small intestines and to a lesser extent in the stomach and colon. This study of the ontogeny of EF shows that the appearance of the protein coincides with the appearance of mature Paneth cells. In new born mouse skin EF is localized in the hair follicles in the first hair cycle from day 2 to day 8. It is however absent in the adult skin. Thus EF is associated with tissues which have a high growth rate.     

Expression of enhancing factor gene and its localization in mouse tissues

Summary Enhancing factor (EF), a 14 kDa protein, isolated from mouse small intestines, has been reported from this laboratory. Based on our earlier studies EF has been implicated in cell proliferation. Preliminary immunohistochemical studies have shown EF to be localized in the Paneth cells of small intestines. In this paper we report the tissue distribution of EF using conditions optimized for immunohistochemical staining. In addition, the data are supported by northern blot analysis using a nick translated cDNA probe specific for EF. The results indicate that EF gene is actively transcribed mainly in the intestines. The chief source of synthesis of EF appears to be the Paneth cells located at the base of the crypts of Lieberkühn.     

Interactions between Canopy Structure and Herbaceous Biomass along Environmental Gradients in Moist Forest and Dry Miombo Woodland of Tanzania

We have limited understanding of how tropical canopy foliage varies along environmental gradients, and how this may in turn affect forest processes and functions. Here, we analyse the relationships between canopy leaf area index (LAI) and above ground herbaceous biomass (AGB_H) along environmental gradients in a moist forest and miombo woodland in Tanzania. We recorded canopy structure and herbaceous biomass in 100 permanent vegetation plots (20 m × 40 m), stratified by elevation. We quantified tree species richness, evenness, Shannon diversity and predominant height as measures of structural variability, and disturbance (tree stumps), soil nutrients and elevation as indicators of environmental variability. Moist forest and miombo woodland differed substantially with respect to nearly all variables tested. Both structural and environmental variables were found to affect LAI and AGB_H, the latter being additionally dependent on LAI in moist forest but not in miombo, where other factors are limiting. Combining structural and environmental predictors yielded the most powerful models. In moist forest, they explained 76% and 25% of deviance in LAI and AGB_H, respectively. In miombo woodland, they explained 82% and 45% of deviance in LAI and AGB_H. In moist forest, LAI increased non-linearly with predominant height and linearly with tree richness, and decreased with soil nitrogen except under high disturbance. Miombo woodland LAI increased linearly with stem density, soil phosphorous and nitrogen, and decreased linearly with tree species evenness. AGB_H in moist forest decreased with LAI at lower elevations whilst increasing slightly at higher elevations. AGB_H in miombo woodland increased linearly with soil nitrogen and soil pH. Overall, moist forest plots had denser canopies and lower AGB_H compared with miombo plots. Further field studies are encouraged, to disentangle the direct influence of LAI on AGB_H from complex interrelationships between stand structure, environmental gradients and disturbance in African forests and woodlands.     

Application of “Streamlined” Material Input per Service Unit Concept to Small Residential Districts in China

This article reports a new application of material and energy accounting techniques to characterize and quantify the relationships between material input (and the related energy flows and emissions) and the services provided (i.e., material input per service unit [MIPS]) at the neighborhood level. The case study focuses on China's small residential district (SRD). It is concluded that linking a service (in this case, residential function) enabled by a given product (neighborhood development) to the amount of materials, energy, and emissions used or produced in creating that product offers a potential way to reduce the environmental impact of that service through more efficient use of materials, enlarged service scales, and improved buying decisions.