VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Solution structure of the major (Spy0128) and minor (Spy0125 and Spy0130) pili subunits from Streptococcus pyogenes

Adhesion of the serotype M1 Streptococcus pyogenes strain SF370 to human tonsil explants and cultured keratinocytes requires extended polymeric surface structures called pili. In this important human pathogen, pili are assembled from three protein subunits: Spy0125, Spy0128 and Spy0130 through the action of sortase enzymes. For this study, the structural properties of these pili proteins have been investigated in solution. Spy0125 and Spy0128 display characteristics of globular, folded proteins. Circular dichroism suggests a largely β-sheet composition for Spy0128 and Spy0125; Spy0130 appears to contain little secondary structure. Each of the proteins adopts a monodisperse, monomeric state in solution as assessed by analytical ultracentrifugation. Further, small-angle X-ray scattering curves for Spy0125, Spy0128 and Spy0130 suggest each protein adopts an elongated shape, likely comprised of two domains, with similar maximal dimensions. Based on the scattering data, dummy atom models of each of the pili subunits have been reconstructed ab initio. This study provides the first insights into the structure of Streptococcus pyogenes minor pili subunits, and possible implications for protein function are discussed.     

Life history characteristics of the clam<Emphasis Type="Italic"> Mya arenaria</Emphasis> in the White Sea

Dynamics of Mya arenaria beds in two bights of the Chupa Inlet (Kandalaksha Bay, White Sea) were studied on a long-term basis. Observations were carried out at 1– to 3-year intervals from 1979 up to 1999. The studied soft-shell clam beds were characterised by a substantial instability of age structure. Since 1988, only one year-class has dominated in the beds while other generations have been scarce and recruitment was not observed. This pattern of Mya bed dynamics was related neither to interannual environmental changes nor to differential reproduction success or predation effects in the benthic assemblages. Favourable conditions for spat formation in 1988 (low abundance of other M. arenaria generations), as well as for juvenile survival during the following winter, resulted in high abundance of juveniles in both investigated locations in 1989. The mortality rate (μ) in this 1988 generation varied throughout the period of investigation and was related to age. The mortality level decreased for the first 2–4 years of the life cycle, then stabilised for the next 3–4 years, and eventually increased in subsequent years. Overall μ values ranged from 0 to 1.68 year^–1. The oldest specimens observed were 17 years old and had a maximum shell length of 79 mm. Significant differences in average growth rates were observed between molluscs of different locations. Communicated by H.-D. Franke     

Translation initiator EIF4G1 mutations in familial Parkinson disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.     

The structure and ligand binding properties of the B. subtilis YkoF gene product, a member of a novel family of thiamin/HMP-binding proteins

The crystal structure of the Bacillus subtilis YkoF gene product, a protein involved in the hydroxymethyl pyrimidine (HMP) salvage pathway, was solved by the multiwavelength anomalous dispersion (MAD) method and refined with data extending to 1.65 A resolution. The atomic model of the protein shows a homodimeric association of two polypeptide chains, each containing an internal repeat of a ferredoxin-like betaalphabetabetaalphabeta fold, as seen in the ACT and RAM-domains. Each repeat shows a remarkable similarity to two members of the COG0011 domain family, the MTH1187 and YBL001c proteins, the crystal structures of which were recently solved by the Northeast Structural Genomics Consortium. Two YkoF monomers form a tightly associated dimer, in which the amino acid residues forming the interface are conserved among family members. A putative small-ligand binding site was located within each repeat in a position analogous to the serine-binding site of the ACT-domain of the Escherichia coli phosphoglycerate dehydrogenase. Genetic data suggested that this could be a thiamin or HMP-binding site. Calorimetric data confirmed that YkoF binds two thiamin molecules with varying affinities and a thiamine-YkoF complex was obtained by co-crystallization. The atomic model of the complex was refined using data to 2.3 A resolution and revealed a unique H-bonding pattern that constitutes the molecular basis of specificity for the HMP moiety of thiamin.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

PROROCENTRUM BELIZEANUM,PROROCENTRUM ELEGANS,AND PROROCENTRUM CARIBBAEUM,THREE NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE1

Three new benthic dinoflagellate species, Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, from mangrove floating detritus are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. belizeanum are round to slightly oval with a cell size of 55–60 μm long and 50–55 μm wide. Areolae are round and numerous (853–1024 per valve) and range from 0.66 to 0.83 μm in size. The periflagellar area of P. belizeanum is a broad V-shaped depression; it accommodates a flagellar and an auxiliary pore and a flared, curved apical collar. The intercalary band of P. belizeanum is horizontally striated. Prorocentrum elegans is a small species 15–20 μm long and 10–14 μm wide, with an ovate cell shape. The thecal surface is smooth. Two sizes of valve pores were recognized: large, round pores (20–22 per valve) arranged in a distinct pattern and smaller pores situated in an array along the intercalary band. The periflagellar area is V-shaped; it accommodates an uneven sized flagellar pore, an auxiliary pore, and an angled protuberant flagellar plate. The intercalary band is transversely striated. It is a bloom-forming species. Prorocentrum caribbaeum cells are heart-shaped with a rounded anterior end and a pointed posterior end. Cells range from 40 to 45 μm long and 30 to 35 μm wide. Thecal surface has two different-sized pores: large, round pores (145–203 per valve) arranged perpendicularly from the posterior margins, and small, round pores unevenly distributed on the thecal surface. The periflagellar area is ornate. It is V-shaped with a curved apical collar located next to the auxiliary pore; a smaller protuberant apical plate is adjacent to the flagellar pore. The intercalary band is transversely striated and sinuous. Cells are active swimmers.