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Background Non-autonomous VNTR (Variable Number of Tandem Repeats) composite retrotransposons – SVA (SINE-R-VNTR-Alu) and LAVA (L1-Alu-VNTR-Alu) – are specific to hominoid primates. SVA expanded in great apes, LAVA in gibbon. Both SVA and LAVA have been shown to be mobilized by the autonomous LINE-1 (L1)-encoded protein machinery in a cell-based assay in trans. The efficiency of human SVA retrotransposition in vitro has, however, been considerably lower than would be expected based on recent pedigree-based in vivo estimates. The VNTR composite elements across hominoids – gibbon LAVA, orangutan SVA_A descendants and hominine SVA_D descendants – display characteristic structures of the 5′ Alu-like domain and the VNTR. Different partner L1 subfamilies are currently active in each of the lineages. The possibility that the lineage-specific types of VNTR composites evolved in response to evolutionary changes in their autonomous partners, particularly in the nucleic acid binding L1 ORF1-encoded protein, has not been addressed. Results Here I report the identification and functional characterization of a highly active human SVA element using an improved mneo retrotransposition reporter cassette. The modified cassette (mneoM) minimizes splicing between the VNTR of human SVAs and the neomycin phosphotransferase stop codon. SVA deletion analysis provides evidence that key elements determining its mobilization efficiency reside in the VNTR and 5′ hexameric repeats. Simultaneous removal of the 5′ hexameric repeats and part of the VNTR has an additive negative effect on mobilization rates. Taking advantage of the modified reporter cassette that facilitates robust cross-species comparison of SVA/LAVA retrotransposition, I show that the ORF1-encoded proteins of the L1 subfamilies currently active in gibbon, orangutan and human do not display substrate preference for gibbon LAVA versus orangutan SVA versus human SVA. Finally, I demonstrate that an orangutan-derived ORF1p supports only limited retrotransposition of SVA/LAVA in trans, despite being fully functional in L1 mobilization in cis. Conclusions Overall, the analysis confirms SVA as a highly active human retrotransposon and preferred substrate of the L1-encoded protein machinery. Based on the results obtained in human cells coevolution of L1 ORF1p and VNTR composites does not appear very likely. The changes in orangutan L1 ORF1p that markedly reduce its mobilization capacity in trans might explain the different SVA insertion rates in the orangutan and hominine lineages, respectively.

SVA (SINE-R-VNTR-Alu) elements are non-autonomous non-LTR (Long Terminal Repeat) retrotransposons. They are found in all hominoid primates but did not amplify to appreciable numbers in gibbons. Recently, phylogenetic networks of hominid (orangutan, gorilla, chimpanzee, human) SVA elements based on comparison of overall sequence identity have been reported. Here I present a detailed phylogeny of SVA_D elements in gorilla, chimpanzee and humans based on sorting of co-segregating substitutions. Complementary comparative genomics analysis revealed that the majority (1763 out of 1826–97%) of SVA_D elements in gorilla represent species-specific insertions – indicating very low activity of the subfamily before the gorilla/chimpanzee-human split. The origin of the human-specific subfamily SVA_F could be traced back to a source element in the hominine common ancestor. The major expanding lineage-specific subfamilies were found to differ between chimpanzee and humans. Precursors of the dominant chimpanzee SVA_D subfamily are present in humans; however, they did not expand to appreciable levels. The analysis also uncovered that one of the chimpanzee-specific subfamilies was formed by splicing of the STK40 first exon to the SVA Alu-like region. Many of the 94 subfamily members contain additional 5′ transductions – among them exons of 8 different other genes. Striking similarities to the MAST2-containing human SVA_F1 suggest parallel evolution of non-canonical SVAs in chimpanzees and humans.

The corticotropin releasing factor (CRF) system plays a critical role in responses to stressful stimuli, and is expressed in many areas of the brain involved in processing fear, anxiety, and social behaviors. To better understand the mechanisms by which the CRF system modulates responses to stressful events and social stimuli, we employed a mouse model that selectively disrupts NMDA receptor function via NMDA receptor subunit NR1 (Grin1) knockout specifically in Cre-expressing CRF neurons. These animals (Cre+/ fGrin1+ ) were compared with littermates lacking Cre expression (Cre−/ fGrin1+ ). Following cue discrimination fear condition- ing, male Cre+/ fGrin1+ mice showed increased fear expression to the tone paired with a foot shock (CS+) while still discriminating the CS+ from a tone never paired with a foot shock (CS−). In contrast to males, female mice learned and discriminated fear cues equivalently across the genotypes. Similarly, no genotype differences in sociability or social novelty were observed in female mice, but Cre+/ fGrin1+ males displayed greater naive sociability and preference for social novelty than Cre−/ fGrin1+ littermates. Furthermore, the level of social withdrawal exhibited by male Cre+/ fGrin1+ mice susceptible to social defeat stress relative to same genotype controls was significantly more pronounced than that displayed by susceptible Cre−/ fGrin1+ mice compared to control Cre−/ fGrin1+ mice. Together, these results demonstrate increased fear, social, and stress responsiveness specifically in male Cre+/ fGrin1+ mice. Our findings indicate that NMDA- mediated glutamatergic regulation of CRF neurons is important for appropriately regulating fear and social responses, likely functioning to promote survival under aversive circumstances. © 2015 Elsevier Ltd. All rights reserved.

Composite retrotransposons are widely distributed in the plant and animal kingdoms. Some of the most complex of these are found in hominoid primates. SVA, LAVA, PVA and FVA combine simple repeats, Alu fragments, a VNTR (Variable Number of Tandem Repeats) and variable 3′ domains, which are, except for PVA, derived from other retrotransposons. Although a likely precursor of SVA–a “tailed VNTR” named SVA2–had been identified in the Rhesus genome, the exact sequence and mechanism of the assembly of this type of composite retrotransposon had been elusive. The discovery of LAVA, PVA and FVA in gibbons provided the opportunity to delineate the order of assembly of the components of VNTR-containing retrotransposons. Our recent analysis suggests that an extinct “Alu-SVA2” acquired variant 3′ ends by splicing. In this commentary I will discuss the mode of assembly of VNTR composites in the context of their capacity to engage in alternative splicing to co-mobilize host RNA sequences and to become exonized. The second part will focus on structural determinants of VNTR composite retrotransposon mobilization in the context of lineage-specific expansion of particular families/subfamilies of these elements.

Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ERalpha (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ERalpha. Downregulation of ERalpha using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ERalpha ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ERalpha ligands. We also detected ERalpha binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ERalpha-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ERalpha-dependent development of malignant diseases.

SINEs (Short INterspersed Elements) are widely distributed among eukaryotes. Some SINE families are organized in superfamilies characterized by a shared central domain. These central domains are conserved across species, classes, and even phyla. Here we report the identification of two novel such superfamilies in the genomes of gastropod and bivalve mollusks. The central conserved domain of the first superfamily is present in SINEs in Caenogastropoda and Vetigastropoda as well as in all four subclasses of Bivalvia. We designated the domain MESC (Romanian for MElc-snail and SCoica-mussel) because it appears to be restricted to snails and mussels. The second superfamily is restricted to Caenogastropoda. Its central conserved domain-Snail-is related to the Nin-DC domain. Furthermore, we provide evidence that a 40-bp subdomain of the SINE V-domain is conserved in SINEs in mollusks and arthropods. It is predicted to form a stable stem-loop structure that is preserved in the context of the overall SINE RNA secondary structure in invertebrates. Our analysis also recovered short retrotransposons with a Long INterspersed Element (LINE)-derived 5' end. These share the body and/or the tail with transfer RNA (tRNA)-derived SINEs within and across species. Finally, we identified CORE SINEs in gastropods and bivalves-extending the distribution range of this superfamily.

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Background VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu), LAVA (LINE-1-Alu-VNTR-Alu), PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu) - are specific to hominoid primates. Their assembly, the evolution of their 5’ and 3’ domains, and the functional significance of the shared 5’ Alu-like region are well understood. The central VNTR domain, by contrast, has long been assumed to represent a more or less random collection of 30-50 bp GC-rich repeats. It is only recently that it attracted attention in the context of regulation of SVA expression. Results Here we provide evidence that the organization of the VNTR is non-random, with conserved repeat unit (RU) arrays at both the 5’ and 3’ ends of the VNTRs of human, chimpanzee and orangutan SVA and gibbon LAVA. The younger SVA subfamilies harbour highly organized internal RU arrays. The composition of these arrays is specific to the human/chimpanzee and orangutan lineages, respectively. Tracing the development of the VNTR through evolution we show for the first time how tandem repeats evolve within the constraints set by a functional, non-autonomous non-LTR retrotransposon in two different families - LAVA and SVA - in different hominoid lineages. Our analysis revealed that a microhomology-driven mechanism mediates expansion/contraction of the VNTR domain at the DNA level. Elements of all four VNTR composite families have been shown to be mobilized by the autonomous LINE1 retrotransposon in trans. In case of SVA, key determinants of mobilization are found in the 5’ hexameric repeat/Alu-like region. We now demonstrate that in LAVA, by contrast, the VNTR domain determines mobilization efficiency in the context of domain swaps between active and inactive elements. Conclusions The central domain of VNTR composites evolves in a lineage-specific manner which gives rise to distinct structures in gibbon LAVA, orangutan SVA, and human/chimpanzee SVA. The differences observed between the families and lineages are likely to have an influence on the expression and mobilization of the elements. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1543-z) contains supplementary material, which is available to authorized users.

The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3'UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3'UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3'UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3' UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.

Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.

SVA (SINE-R-VNTR-Alu) elements constitute the youngest family of composite non-LTR retrotransposons in hominoid primates. The sequence of their assembly, however, remains unclear. Recently, a second family of VNTR-containing composites, LAVA (L1-Alu-VNTR-Alu), has been identified in gibbons. We now report the existence of two additional VNTR composite families, PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu), in the genome of Nomascus leucogenys. Like LAVA, they share the 5'-Alu-like region and VNTR with SVA, but differ at their 3'-ends. The 3'-end of PVA comprises part of the PTGR2 gene, whereas FVA is characterized by the presence of a partial FRAM element in its 3'-domain. Splicing could be identified as the mechanism of acquisition of the variant 3'-ends in all four families of VNTR composites. SVAs have been shown to be mobilized by the L1 protein machinery in trans. A critical role in this process has been ascribed to their 5'-hexameric repeat/ Alu-like region. The Alu-like region displays specific features in each of the VNTR composite families/subfamilies with characteristic deletions found in the evolutionary younger subfamilies. Using reciprocal exchanges between SVA_E and PVA/FVA elements, we demonstrate that the structure, not the presence of the (CCCTCT)n/ Alu-like region determines mobilization capacity. Combination of LAVA and SVA_E domains does not yield any active elements-suggesting the use of different combinations of host factors for the two major groups of VNTR composites. Finally, we demonstrate that the LAVA 3'-L1ME5 fragment attenuates mobilization capacity.

Carbone, Lucia et al.-- This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported licence., Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

Sequencing of rat and human vascular endothelial growth factor (VEGF) cDNA clones has previously identified a 3' untranslated region of approximately 1.9 kb, although the apparent site of polyadenylation differed in the two species, despite a high degree of sequence conservation in the region. Neither site is preceded by a canonical AAUAAA polyadenylation signal, a situation frequently found in genes that are subject to alternative polyadenylation. We have sequenced 2.25 kb of the 3' region of the mouse VEGF gene and mapped the usage of potential polyadenylation sites in fibroblasts cultured under both normoxic and hypoxic conditions. We find that two sites for polyadenylation are present in the mouse VEGF gene but the majority of transcripts contain the longer form of the 3'UTR and that their usage is not effected by environmental oxygen tension.

The endothelial cell-specific mitogen vascular-endothelial growth factor (VEGF) plays a key role in both physiological and pathological angiogenesis. The up-regulation of VEGF expression in response to reduced oxygen tension occurs through transcriptional and post-transcriptional mechanisms. To investigate the molecular mechanisms of transcriptional activation by hypoxia (1% oxygen), fine mapping of a hypoxia-responsive region of the human VEGF promoter was carried out using luciferase reporter-gene constructs in C6 glioma cells. Here, we report that the binding site of hypoxia-inducible factor 1 (HIF1) is crucial for the hypoxic induction of VEGF gene expression. However, an enhancer subfragment containing the HIF1 binding site was not sufficient to confer full hypoxia responsiveness. Addition of upstream sequences restored the full sensitivity to hypoxia induction. This potentiating effect is due to activator protein 1 binding. The ‘potentiating’ sequences are unable to confer hypoxia responsiveness on their own. Our results strongly suggest that in C6 glioma cells a complex array of trans-acting factors facilitates full transcriptional induction of VEGF gene expression by hypoxia.

Most common methods for inferring transposable element (TE) evolutionary relationships are based on dividing TEs into subfamilies using shared diagnostic nucleotides. Although originally justified based on the “master gene” model of TE evolution, computational and experimental work indicates that many of the subfamilies generated by these methods contain multiple source elements. This implies that subfamily-based methods give an incomplete picture of TE relationships. Studies on selection, functional exaptation, and predictions of horizontal transfer may all be affected. Here, we develop a Bayesian method for inferring TE ancestry that gives the probability that each sequence was replicative, its frequency of replication, and the probability that each extant TE sequence came from each possible ancestral sequence. Applying our method to 986 members of the newly-discovered LAVA family of TEs, we show that there were far more source elements in the history of LAVA expansion than subfamilies identified using the CoSeg subfamily-classification program. We also identify multiple replicative elements in the AluSc subfamily in humans. Our results strongly indicate that a reassessment of subfamily structures is necessary to obtain accurate estimates of mutation processes, phylogenetic relationships and historical times of activity., Author Summary The most common entities in vertebrate genomes are transposable elements (TEs), DNA sequences that have been repeatedly copied and inserted into new locations throughout the genome. Some TEs have been replicated hundreds of thousands of times, and their ecology and evolutionary history within a genome is thus critical to understanding how genome structure evolves. It was once thought that only a few “master gene” copies could replicate, while the rest were inactive (dead on arrival), but recent computational and laboratory studies have indicated that this is not the case. However, previous methods for reconstructing TE evolutionary history were not designed to solve the problem of determining the ancestral source sequence for large numbers of elements. Here, we present a new method that is. Our method surveys all likely TE ancestors and determines the probability that each modern element arose from each of its plausible ancestors. We applied our method to the gibbon-derived LAVA TE family and to the human AluSc subfamily and inferred many more source elements than indicated by previous methods. This new method will help us better understand TE evolution, including both the impact of sequence on replication and the substitution process after replication.

Since the short intron in the 5′-untranslated region (5′-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5′-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5′-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins1IVS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides +87 and +110 showed a reduction of expression by 30 %. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Size distributions of extracts derived from starch were investigated to aid in elucidating structure-function relationships of these polymers in water. Starch granules derived from waxy maize and amylomaize VII were dissolved in water by microwave heating in a high pressure vessel. Transmission electron microscopy of starch deposited from dilute solution and rotary shadowed with platinum, revealed that amylopectin imaged from waxy maize could be broadly classified as about 28% circular space filling patches containing branched clusters and 72% asymmetric linear containing branched clusters. Lengths of asymmetric linear amylopectin components ranged from about 37 to 980 nm whereas the diameter of circular amylopectin components ranged from about 44 to 200 nm. Although the starch in amylomaize VII is about 70% amylose, its narrow asymmetric structure when visualized by microscopy enabled us to image amylose even though amylopectin was present. Lengths of components ranged from about 46 to 254 nm. After smoothing and curve fitting, we found that all size distributions investigated could be treated as if they were multimodal in nature. The most abundant amylose component had a linear density of 8.2 × 103 molar mass units/ nm. This value could be explained if amylose had an aggregation number of about 5.9.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.

SVA elements are nonautonomous non–long terminal repeat (non-LTR) retrotransposons that originated

During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tal1 transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegflo/lo embryos carrying a hypomorph allele, Flk1-positive cells reach the yolk sac at E8.5,but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development,different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegflo/lo embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Vegflo allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 andβH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment.

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegflo. Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing ‘Vegf wild-type’ visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the ‘Vegf wild-type’ yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murineFlk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5′-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5′-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

Vascular endothelial growth factor (VEGF), an essential regulator of angiogenesis during early development as well as during the growth of solid tumours, bears an unusually large 5′ untranslated region (5′-UTR) in the mRNA of over 1000 nucleotides. We found that the VEGF 5′-UTR, despite being GC-rich and containing an upstream short open reading frame, promotes efficient translation of a luciferase reporter. The VEGF 5′-UTR also allowed translation of luciferase from a dicistronic mRNA when placed between the two cistrons, demonstrating that it contains an internal ribosome entry site. Deletion analysis indicated that the IRES resides towards the 3′ end of the 5′-UTR.

Angiogenesis, the sprouting of capillaries from pre-existing vessels, is of fundamental importance during embryonic vascular development and during certain physiological processes in the adult organism, for example menstruation, pregnancy, or wound healing. Moreover, angiogenesis plays a major role in the pathogenesis of several diseases, such as proliferative retinopathy, psoriasis, or solid tumor growth (Folkman, 1995). The growth of solid tumors is largely dependent on the supply of oxygen and nutrients from the blood stream. Small tumors of less than 1–2 mm in diameter are not vascularized, and they can be nourished by simple diffusion. Such avascular tumors do not induce neovascularization, and they rarely metastasize. However, further tumor growth is dependent on a vascular network that is able to fulfill the demands of the growing tumor for oxygen and nutrients. Vascularized tumors induce host vessels to extend vascular sprouts. They have the potential to expand their cell population, and they may eventually metastasize (for review, see Folkman, 1995).

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

The development of effective quantitative microbial risk-assessment models for foodborne pathogens depends on the availability of data on the consumers' exposure to a biological agent and the dose-response relationship that relates levels of the biological agent ingested with frequency of infection or disease. Information on the latter has historically been acquired from human volunteer feeding studies. However, such studies are not feasible for pathogens that either have a significant risk of being life threatening or for which morbidity is primarily associated with high-risk populations (i.e., immunocompromised persons). For these pathogens, it is proposed that purposefully conservative dose-response relationships can be estimated on the basis of combining available epidemiologic data with food-survey data for a ready-to-eat product. As an example, data on the incidence of listeriosis in Germany were combined with data on the levels of Listeria monocytogenes in smoked fish to generate a dose-response curve for this foodborne pathogen.

To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system.

Antibody titers against Candida albicans and Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, were determined in normal subjects, and in patients with tinea versicolor or chronic mucocutaneous candidiasis. Whereas antibody against both organisms was found in low titer in normal subjects, a majority of patients with each infection had elevated antibody titers against the infecting organism. Patients infected with one organism did not have elevated titers against the other, and titers against one did not correlate with titers against the other. Therefore, these studies indicate that both of the superficial infections are capable of inducing a significant humoral immune response and that actual infection with the organism rather than mere colonization is required for production of the elevated antibody titers.

Cephalothin significantly suppressed in vitro DNA and total protein synthesis in human peripheral blood lymphocytes stimulated by antigens or mitogens. However, similar concentrations of this antibiotic enhanced streptokinase-streptodornase-stimulated production of the lymphokine leukocyte migration-inhibition factor (LMIF) and directly stimulated production of this lymphokine by otherwise unstimulated lymphocytes from 10 of 12 normal human subjects. Penicillin did not appear to produce these effects. Cephalothin did not interfere directly with neutrophil migration or the interaction of preformed LMIF with neutrophils. Stimulation of LMIF production by cephalothin required viable lymphocytes and was inhibited by puromycin. These results suggest that cephalothin is capable of inducing lymphokine production by human lymphocytes in a manner that appears to be nonspecific in nature. This type of effect could be the basis of some apparently immunologic reactions to this antibiotic.

The effect of chloramphenicol on the in vitro function of human peripheral blood lymphocytes was studied in assays of lymphocyte transformation and lymphokine production. When lymphocytes were stimulated by phytohemagglutinin, concanavalin A, or pokeweed mitogen in the presence of various concentrations of chloramphenicol, only minimal effects on blastogenesis were noted. However, suppression by chloramphenicol of blastogenesis induced by candida antigen or streptokinase-streptodornase was greater in magnitude and was dose-dependent; blastogenesis was suppressed to 25%--30% or normal levels by concentrations of chloramphenicol of 25--50 microgram/ml. Chloramphenicol had little effect on the production of the lymphokine leukocyte migration inhibition factor by lymphocytes stimulated either by candida antigen or by concanavalin A, whereas puromycin at a concentration of 5 microgram/ml significantly suppressed this response. Thus chloramphenicol appears to suppress antigen-induced lymphocyte blastogenesis significantly but not lymphokine production by stimulated lymphocytes.

Angiogenic growth factors and their endothelial receptors are thought to function as major regulators of blood vessel formation. Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2), as well as Angiopoietin-1 and its receptor, Tie-2, represent key signal transduction systems involved in the regulation of embryonic vascular development. The expression of these molecules correlates with phases of blood vessel formation during embryogenesis. Inactivation of any of the genes encoding these molecules in mouse embryos results in defective vascular development and embryonic lethality around mid-gestation. In addition, the VEGF signal transduction system has been implicated in the regulation of pathological blood vessel growth during certain angiogenesis-dependent diseases that are often associated with tissue ischemia, such as proliferative retinopathy or solid tumor growth. This hypothesis is substantiated by experiments, in which the inhibition of VEGF signal transduction resulted in the the inhibition of neovascularization in these diseases. Thus, the VEGF signal transduction system represents a useful target for an anti-angiogenic therapy.