Serpin-derived peptides are antiangiogenic and suppress breast tumor xenograft growth

Angiogenesis is the formation of neovasculature from preexisting microvessels. Several endogenous proteins regulate the balance of vessel formation and regression in the body including pigment epithelium-derived factor (PEDF), which has been shown to be antiangiogenic and to suppress tumor growth. Using sequence homology and bioinformatics, we previously identified several peptide sequences homologous to an active region of PEDF existing in multiple proteins in the human proteome. These short 11-mer peptides are found in a DEAH box helicase protein, CKIP-1 and caspase 10, and show similar activity in altering endothelial cell adhesion, migration and inducing apoptosis.We tested the peptide derived from DEAH box helicase protein in a triple-negative MDA-MB-231 breast orthotopic xenograft model in severe combined immunodeficient mice and show significant tumor suppression.     

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.     

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.     

Phospholipid-induced structural changes to an erythroid beta spectrin ankyrin-dependent lipid-binding site

The region of beta-spectrin that is responsible for interactions with ankyrin was shown to comprise an ankyrin-sensitive lipid-binding site. Structural studies indicate that it exhibits a mixed 3(10)/alpha helical conformation and is highly amphipathic. These features together with the distinctively conserved sequence of the lipid-binding site motivated us to explore the mechanism of its interactions with biological membranes. A series of singly and doubly spin-labeled erythroid beta-spectrin-derived peptides was constructed, and the spin-label mobility and spin-spin distances were analyzed via electron paramagnetic resonance spectroscopy and two different calculation methods. The results indicate that in beta-spectrin, the lipid-binding domain, which is part of the 14(th) segment, has the topology of typical triple-helical spectrin repeat. However, it undergoes significant changes when interacting with phospholipids or detergents. A mechanism for these interactions is proposed in this paper.     

Heightened aberrant deposition of hard-wearing elastin in conduit arteries of prehypertensive SHR is associated with increased stiffness and inward remodeling

Elastin is a major component of conduit arteries and a key determinant of vascular viscoelastic properties. Aberrant organization of elastic lamellae has been reported in resistance vessels from spontaneously hypertensive rats (SHR) before the development of hypertension. Hence, we have characterized the content and organization of elastic lamellae in conduit vessels of neonatal SHR in detail, comparing the carotid arteries from 1-wk-old SHR with those from Wistar-Kyoto (WKY) and Sprague Dawley (SD) rats. The general structure and mechanics were studied by pressure myography, and the internal elastic lamina organization was determined by confocal microscopy. Cyanide bromide-insoluble elastin scaffolds were also prepared from 1-mo-old SHR and WKY aortas to assess their weight, amino acid composition, three-dimensional lamellar organization, and mechanical characteristics. Carotid arteries from 1-wk-old SHR exhibited narrower lumen and greater intrinsic stiffness than those from their WKY and SD counterparts. These aberrations were associated with heightened elastin content and with a striking reduction in the size of the fenestrae present in the elastic lamellae. The elastin scaffolds isolated from SHR aortas also exhibited increased relative weight and stiffness, as well as the presence of peculiar trabeculae inside the fenestra that reduced their size. We suggest that the excessive and aberrant elastin deposited in SHR vessels during perinatal development alters their mechanical properties. Such abnormalities are likely to compromise vessel expansion during a critical period of growth and, at later stages, they could compromise hemodynamic function and participate in the development of systemic hypertension.     

Selection of the conodont Idiognathodus simulator (Ellison) as the event marker for the base of the global Gzhelian Stage (Upper Pennsylvanian, Carboniferous)

Studies carried out for more than 10 years by the Task Group to establish GSSPs at the base of the Moscovian–Kasimovian and Kasimovian–Gzhelian boundaries have resulted in the proposal that the level at which the conodont species Idiognathodus simulator (Ellison, 1941) first appears be selected to mark the base of the Gzhelian Stage. This expands this eastern European chronostratigraphic unit to a global scale.I. simulator (sensu Barrick et al., 2008) has been identified so far in Midcontinent and eastern North America, the Moscow and Donets basins and southern Urals of eastern Europe, and in south-central China. Correlation of this level based on this species and other conodont species can be reinforced in some areas by ammonoid and fusulinid data.     

Locus-aware decomposition of gene trees with respect to polytomous species trees

Background

Horizontal gene transfer (HGT), a process of acquisition and fixation of foreign genetic material, is an important biological phenomenon. Several approaches to HGT inference have been proposed. However, most of them either rely on approximate, non-phylogenetic methods or on the tree reconciliation, which is computationally intensive and sensitive to parameter values.

Results

We investigate the locus tree inference problem as a possible alternative that combines the advantages of both approaches. We present several algorithms to solve the problem in the parsimony framework. We introduce a novel tree mapping, which allows us to obtain a heuristic solution to the problems of locus tree inference and duplication classification.

Conclusions

Our approach allows for faster comparisons of gene and species trees and improves known algorithms for duplication inference in the presence of polytomies in the species trees. We have implemented our algorithms in a software tool available at https://github.com/mciach/LocusTreeInference.