Nuclear small-subunit ribosomal RNA gene-based characterization, molecular phylogeny and PCR detection of the Neoparamoeba from western Long Island Sound lobster

Author Posting. © National Shellfisheries Association, 2005. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 24 (2005): 719-731, doi:10.2983/0730-8000(2005)24[719:NSRRGC]2.0.CO;2.Western Long Island Sound (LIS) lobsters collected by trawl surveys, lobstermen and coastal residents during 2000 to 2002 were identified histologically as infected with a parasome-containing amoeba. Primers to conserved SSU rRNA sequences of parasome-containing amoebae and their nonparasome-containing relatives were used to amplify overlapping SSU rRNA fragments of the presumptive parasite from gill, antenna, antennal gland and ventral nerve cord of infected lobsters. The consensus sequence constructed from these fragments had 98% or greater nucleotide sequence identity with SSU rRNA gene sequences of strains of Neoparamoeba pemaquidensis and associated with high confidence in distance- and parsimony-based phylogenetic analyses with strains of Neoparamoeba pemaquidensis and not members of the family Paramoebidae, e.g., Paramoeba eilhardi. Primers designed to SSU rRNA sequences of the lobster amoeba and other paramoebid/vexilliferid amoebae were used in a nested polymerase chain reaction (PCR) protocol to test DNA extracted from formalin-fixed paraffin-embedded tissues of lobsters collected during the 1999 die-off, when this amoeba initially was identified by light and electron microscopy and reported to be a paramoeba of the genera Paramoeba or Neoparamoeba (Mullen et al. 2004). All sequences amplified from 1999 lobsters, with the exception of one, had 98% to 99% identity to each other, and the 1999 PCR product consensus had 98% identity to Neoparamoeba pemaquidensis strains CCAP 1560/4 (AF371969.1) and 1560/5 (AF371970.1). Molecular characterization of the amoeba from western LIS lobsters by direct amplification circumvents a collective inability to culture the organism in vitro, provides insight into the molecular epidemiology of neoparamoebiasis in American lobster, and allows for PCR-based detection of infected lobsters for future research and diagnostics.Funding for this work was provided by the Connecticut Department of Environmental Protection under Long Island Sound Research Fund Grant No. CWF 333-R to S. Frasca; and by the Connecticut Sea Grant College Program, Grants No. LR/LR-4 to R. Gast and No. LR/LR-5 to P. Gillevet and C. O’Kelly, through the US Department of Commerce, National Oceanic and Atmospheric Administration (NOAA), Award NA16RG1364

Origin licensing and p53 status regulate Cdk2 activity during G1

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G1/S transition to prevent rereplication. The strict inhibition of licensing after the G1/S transition means that all origins used in S phase must have been licensed in the preceding G1. Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G1 progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells

Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment

Defective Cell Cycle Checkpoint Functions in Melanoma Are Associated with Altered Patterns of Gene Expression

Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression

Chamber identity programs drive early functional partitioning of the heart

The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction

Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels

Regulated vascular endothelial growth factor (VEGF) signaling is required for proper angiogenesis, and excess VEGF signaling results in aberrantly formed vessels that do not function properly. Tumor endothelial cells have excess centrosomes and are aneuploid, properties that probably contribute to the morphologic and functional abnormalities of tumor vessels. We hypothesized that endothelial cell centrosome number is regulated by signaling via angiogenic factors, such as VEGF. We found that endothelial cells in developing vessels exposed to elevated VEGF signaling display centrosome overduplication. Signaling from VEGF, through either MEK/ERK or AKT to cyclin E/Cdk2, is amplified in association with centrosome overduplication, and blockade of relevant pathway components rescued the centrosome overduplication defect. Endothelial cells exposed to elevated FGF also had excess centrosomes, suggesting that multiple angiogenic factors regulate centrosome number. Endothelial cells with excess centrosomes survived and formed aberrant spindles at mitosis. Developing vessels exposed to elevated VEGF signaling also exhibited increased aneuploidy of endothelial cells, which is associated with cellular dysfunction. These results provide the first link between VEGF signaling and regulation of the centrosome duplication cycle, and suggest that endothelial cell centrosome overduplication contributes to aberrant angiogenesis in developing vessel networks exposed to excess angiogenic factors

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment