Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.     

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.     

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.     

Spatio-temporal filtering properties of a dendritic cable with active spines: A modeling study in the spike-diffuse-spike framework

The spike-diffuse-spike (SDS) model describes a passive dendritic tree with active dendritic spines. Spine-head dynamics is modeled with a simple integrate-and-fire process, whilst communication between spines is mediated by the cable equation. In this paper we develop a computational framework that allows the study of multiple spiking events in a network of such spines embedded on a simple one-dimensional cable. In the first instance this system is shown to support saltatory waves with the same qualitative features as those observed in a model with Hodgkin-Huxley kinetics in the spine-head. Moreover, there is excellent agreement with the analytically calculated speed for a solitary saltatory pulse. Upon driving the system with time-varying external input we find that the distribution of spines can play a crucial role in determining spatio-temporal filtering properties. In particular, the SDS model in response to periodic pulse train shows a positive correlation between spine density and low-pass temporal filtering that is consistent with the experimental results of Rose and Fortune [1999, ‘Mechanisms for generating temporal filters in the electrosensory system,’ The Journal of Experimental Biology 202: 1281–1289]. Further, we demonstrate the robustness of observed wave properties to natural sources of noise that arise both in the cable and the spine-head, and highlight the possibility of purely noise induced waves and coherent oscillations. Action Editor: Erik De Schutter     

Anthropogenic scarring of western gray whales (Eschrichtius robustus)

Western gray whales ( Eschrichtius robustus ) are critically endangered and anthropogenic threats, such as entanglement in fishing gear and collisions with vessels, may be acting to limit recovery of the population. Thus, examining the magnitude of such anthropogenic interactions using a scar-based approach is warranted. A multi-year (1995–2005) photo-identification study of western gray whales on their feeding ground off northeastern Sakhalin Island, Russia, has resulted in a large data set of digital and film images of 150 individuals. These images were reviewed and scored for anthropogenic scarring by recording the presence of visible scars resulting from fishing gear entanglement and vessel collisions in 21 defined body regions. In total, 20.0% ( n = 30) of whales identified during the study period had detectable anthropogenic scarring, with 18.7% ( n = 28) determined to have been previously entangled in fishing gear at least once and 2.0% ( n = 3) to have survived at least one vessel collision. These estimates are likely to be conservative given the nature of the photo-identification data set, but indicate that male and female western gray whales are subject to anthropogenic interactions. Future studies designed to systematically estimate the frequency and rates of anthropogenic events are needed and would have direct conservation and management implications.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.     

Mammalian end binding proteins control persistent microtubule growth

End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.     

Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of radial growth, tree age, climate, and seed origin on wood density of diverse jack pine populations

Several models of the effects of silviculture, radial growth, and tree age on wood density have been developed, but they have rarely considered the roles of diverse seed origins and climate. We developed a model to test the effects of radial growth, tree age, climate, and seed-source origins on wood density in 21 diverse populations of jack pine in a common garden in Petawawa, Ontario, Canada over the last 24 years using a linear mixed-effects model. Although we found significant differences in wood density among diverse seed origins, there were no differences between seed origins having the same ring age and ring width, indicating an indirect effect on wood density of seed-source origin via radial growth. High variation in wood density among trees within the same population and between populations indicated high genetic control of wood density. The climate effect was significant on wood density in all populations, but smaller when radial growth was controlled. Climate effect did not differ significantly among populations. Precipitation in July negatively affected latewood density, whereas precipitation in May in the current year and September of the previous year negatively affected earlywood density. We concluded that a single model of jack pine wood density and radial growth could be used, either controlling for climate effects or not, as the relationship between wood density and radial growth is preserved among the diverse populations, and the climate effect controlling for radial growth in the model was only slight.