An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.     

Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Here protein synthesis is traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon.     

Atractis marquezi n. sp. (Nematoda: Atractidae) and a revision of Atractis dujardin, 1845, sensu Baker, 1987

Atractis marquezi n. sp. from the large intestine of the tortoise Geochelone nigrita is described and illustrated. Atractis Dujardin, 1845, sensu Baker, 1987, is revised to contain only those atractids having a lagonoid spicule. The remaining species of Atractis sensu Baker, 1987, are assigned to Cyrtosomum, Pseudatractis, and Klossinemella. A. marquezi represents the 12th species to be assigned to this genus and is distinguished from other Neotropical species by the distribution pattern of caudal papillae of the male: 2 pairs precloacal, 2 pairs adcloacal, and 6 pairs postcloacal.     

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.     

The stable carbon and nitrogen isotopic composition of vegetation in tropical forests of the Amazon Basin, Brazil

Here we present the within-site, seasonal, and interannual variations of the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios of leaves, wood, bark and litter from four sites in the Amazon region, Brazil. Samples were collected in Manaus (3° 06′07′′ S; 60°01′30′′ W), Ji-Paraná (10°53′07′′ S; 61°57′06′′ W), and Santarém (2°26′35′′ S; 54°42′30′′ W) with mean annual precipitation of 2207, 2040 and 1909 mm respectively. The overall average for all leaf samples was for δ¹³C and for δ¹⁵N (n=756). The leaf δ values at these sites were often but not always statistically distinct from each other. The δ¹³C values varied from to . Pronounced differences in δ¹³C values occurred with height associated with differences in forest structure. The δ¹³C of leaf dry matter showed seasonal variations associated with the length of the dry season, despite the fact that total annual precipitation was similar among the studied sites. Leaf δ¹⁵N values ranged from to a maximum value of , and the Santarém sites showed more enriched values than Manaus and Ji-Paraná sites. No seasonal variation was detected in the δ¹⁵N of leaves, but significant differences were observed among sites and with changes in canopy height. The isotope ratio data are consistent with our current understanding of the roles of light, water availability, and recycling of soil-respired CO₂ influences on δ¹³C and consistent with our understanding that an open nitrogen cycle can lead to high δ¹⁵N values despite a significant number of legumes in the vegetation.     

Phylogenetic Analysis of Heterothallic Neurospora Species

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.     

Treatment with apolipoprotein A-1 mimetic peptide reduces lupus-like manifestations in a murine lupus model of accelerated atherosclerosis

Introduction

The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE^-/-Fas^-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.

Methods

Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.

Results

In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (P_{L, LP} < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (P_L < 0.05) and oxidized phospholipids (oxPLs) (P_{L, LP} < 0.005), and elevated total and vertebral bone mineral density (P_{L, LP} < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68⁺ macrophages (P_LP < 0.01), significantly increased mean α-actin stained area (P_LP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (P_{L, LP} < 0.0005) and VCAM-1 (P_L < 0.0002).

Conclusions

L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis.