Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

Effects of ursolic acid on different steps of the angiogenic process

Ursolic acid is a triterpenoid with pleiotropic biological effects. In this report, we study the effects of ursolic acid on different key steps of angiogenesis. Our results show that ursolic acid is able to inhibit key steps of angiogenesis in vitro, including endothelial cell proliferation, migration, and differentiation. At the same time, it seems to stimulate other key steps of angiogenesis, such as extracellular matrix degradation by MMP-2 and urokinase. Although ursolic acid can inhibit in vivo angiogenesis in the CAM assay, the different signs of the effects it causes on different steps of angiogenesis force one to be cautious concerning its anti-angiogenic potential.     

The fat-like gene of Drosophila is the true orthologue of vertebrate fat cadherins and is involved in the formation of tubular organs

Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. In this paper, we describe the second fat cadherin in Drosophila, fat-like (ftl). We show that ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on our results, we propose a model where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily.     

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.     

Cytotoxic T-lymphocyte escape does not always explain the transient control of simian immunodeficiency virus SIVmac239 viremia in adenovirus-boosted and DNA-primed Mamu-A*01-positive rhesus macaques

Adenovirus 5 (Ad5) vectors show promise as human immunodeficiency virus vaccine candidates. Indian rhesus macaques vaccinated with Ad5-gag controlled simian-human immunodeficiency virus SHIV89.6P viral replication in the absence of Env immunogens that might elicit humoral immunity. Here we immunized 15 macaques using either a homologous Ad5-gag/Ad5-gag (Ad5/Ad5) or a heterologous DNA-gag/Ad5-gag (DNA/Ad5) prime-boost regimen and challenged them with a high dose of simian immunodeficiency virus SIVmac239. Macaques vaccinated with the DNA/Ad5 regimen experienced a brief viral load nadir of less than 10,000 viral copies per ml blood plasma that was not seen in Mamu-A*01-negative DNA/Ad5 vaccinees, Mamu-A*01-positive Ad5/Ad5 vaccinees, or vaccine-naive controls. Interestingly, most of these animals were not durably protected from disease progression when challenged with SIVmac239. To investigate the reasons underlying this short-lived vaccine effect, we investigated breadth of the T-cell response, immunogenetic background, and viral escape from CD8+ lymphocytes that recognize immunodominant T-cell epitopes. We show that these animals do not mount unusually broad cellular immune response, nor do they express unusual major histocompatibility complex class I alleles. Viral recrudescence occurred in four of the five Mamu-A*01-positive vaccinated macaques. However, only a single animal in this group demonstrated viral escape in the immunodominant Gag181-189 CM9 response. These results suggest that viral "breakthrough" in vaccinated animals and viral escape are not inextricably linked and underscore the need for additional research into the mechanisms of vaccine failure.