VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

Bicaudal‐D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule‐based motors is essential for cell organisation and function. The Bicaudal‐D (BicD) protein participates in the transport of a subset of cargoes by the minus‐end‐directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co‐precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin‐mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high‐frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin‐associated trafficking processes and show a novel requirement for microtubule‐based motor transport in the synaptic vesicle cycle.     

Phytoplankton dynamics in a deep, tropical, hyposaline lake

The annual variation of the phytoplankton assemblage of deep (64.6 m), hyposaline (8.5 g l^–1) Lake Alchichica, central Mexico (19 ° N, 97° W), was analyzed in relation to thermal regime, and nutrients concentrations. Lake Alchichica is warm monomictic with a 3-month circulation period during the dry, cold season. During the stratified period in the warm, wet season, the hypolimnion became anoxic. N–NH₃ ranged between non detectable (n.d.) and 0.98 mg l^–1, N–NO₂ between n.d. and 0.007 mg l^–1, N–NO₃ from 0.1 to 1.0 mg l^–1 and P–PO₄ from n.d. to 0.54 mg l^–1. Highest nutrient concentrations were found in the circulation period. Chlorophyll a varied from <1 to 19.8 g l^–1 but most values were <5 g l^–1. The euphotic zone (>1% PAR) usually comprised the top 15–20 m. Nineteen algae species were identified, most of them are typical inhabitants of salt lakes. Diatoms showed the highest species number (10) but the small chlorophyte Monoraphidium minutum, the single-cell cyanobacteria, Synechocystis aquatilis, and the colonial chlorophyte, Oocystis parva, were the numerical dominant species over the annual cycle. Chlorophytes, small cyanobacteria and diatoms dominated in the circulation period producing a bloom comparable to the spring bloom in temperate lakes. At the end of the circulation and at the beginning of stratification periods, the presence of a bloom of the nitrogen-fixing cyanobacteria, N. spumigena, indicated nitrogen-deficit conditions. The well-stratified season was characterized by low epilimnetic nutrients levels and the dominance of small single-cell cyanobacteria and colonial chlorophytes. Phytoplankton dynamics in tropical Lake Alchichica is similar to the pattern observed in some deep, hyposaline, North American temperate lakes.     

Late Pleistocene and Holocene pollen stratigraphy at Lago di Vico, central Italy

A new pollen record from Lago di Vico (core V1) provides fundamental new information towards reconstruction of flora and vegetation history in central Italy during the last 90 000 years. The chronological framework is secured by seventeen AMS¹⁴C dates, one⁴⁰Ar/³⁹Ar date and tephra analyses. At the base of the pollen record, i.e. shortly after the⁴⁰Ar/³⁹Ar date 87 000±7000 B.P., three phases with significant expansion of trees are recorded in close succession. These forest phases, which stratigraphically correspond to St Germain II (and Ognon?) and precede pleniglacial steppe vegetation, are designated by the local names Etruria I, Etruria II and Etruria III. During the pleniglacial, a number of fluctuations of angiosperm mesophilous trees suggest the presence of tree refugia in the area. The lowest pollen concentration values are recorded at ca. 22 000 B.P. which corresponds with other pollen records from the region. The late-glacial is characterized by an expansion in the arboreal pollen curves that is less pronounced, however, than in other pollen profiles from Italy. The Holocene part of the profile is consistently dominated by deciduous oak pollen. No major changes in arboreal pollen composition are recorded but several marked and sudden declines of the tree pollen concentration suggest that the forest cover underwent dramatic changes. Clear evidence for human impact is recorded only when cultivated crops became important which dates to ca. 2630±95 B.P.     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

Differential accumulation of soluble amyloid β peptides 1–40 and 1–42 in human monocytic and neuroblastoma cell lines

Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid beta protein (A(beta)). While A(beta)1-40 predominates in the vascular system, A(beta)1-42 is the major component of the senile plaques in the neuropil. The concentration of both A(beta) species required to form amyloid fibrils in vitro is micromolar, yet soluble A(betas) found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of A(beta) sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of A(beta), we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125I-A(beta)1-40 and 125I-A(beta)1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of A(beta) in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125I-A(betas) than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125I-A(beta)1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125I-A(beta)1-40. These results support that A(beta)1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar A(beta)1-42, we propose that intracellular amyloidogenesis in microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease.     

Naupliar growth versus egg production in the calanoid copepod Centropages typicus

Growth rates of the copepod Centropages typicus were assessedduring May 1998 in the Alboran Sea. The goal of the work wasto compare field growth rates of juvenile (artificial cohortmethod) and adult copepods (female egg production rates) underan extensive range of natural conditions. The results showedthat adult and juvenile growth rates were similar in some stations,while in others no such relationship was apparent. In contrastto the common belief that juvenile growth may be saturated undernatural conditions whereas adults may be food-limited, in mostof our study, adult growth rates were greater than juveniles.We discuss these results in the light of food availability atthe surveyed stations. In summary, the assumption that copepodgrowth rates estimated through egg production rates of adultsare equivalent to juvenile growth is not always valid.     

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)_n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)_n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.     

Ligation activity of fragmented ribozymes in frozen solution: implications for the RNA world

A vexing difficulty of the RNA world hypothesis is how RNA molecules of significant complexity could ever have evolved given their susceptibility to degradation. One way degradation might have been reduced is through low temperature. Here we report that truncated and fragmented derivatives of the hairpin ribozyme can catalyze ligation of a wide variety of RNA molecules to a given sequence in frozen solution despite having little or no activity under standard solution conditions. These results suggest that complex RNAs could have evolved in freezing environments on the early earth and perhaps elsewhere.