Attenuation of TGF-β signaling suppresses premature senescence in a p21-dependent manner and promotes oncogenic Ras-mediated metastatic transformation in human mammary epithelial cells

The molecular mechanisms that drive triple-negative, basal-like breast cancer progression are elusive. Few molecular targets have been identified for the prevention or treatment of this disease. Here we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered transforming growth factor-β (TGF-β) sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative type II receptor (DNRII) of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-Ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, coexpression of DNRII and H-Ras-V12 rendered HMECs highly tumorigenic and metastatic in vivo in comparison with H-Ras-V12-transformed HMECs that spontaneously escaped H-Ras-V12-induced SLGA. Microarray analysis revealed that p21 was the major player mediating Ras-induced SLGA, and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling was blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-Ras-V12-induced SLGA. Our results identify that autocrine TGF-β signaling is an integral part of the cellular anti-transformation network by suppressing the expression of a host of genes, including p21-regulated genes, that mediate oncogene-induced transformation in basal-like breast cancer.     

Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.     

Asymptotically exact analysis of stochastic metapopulation dynamics with explicit spatial structure

We describe a mathematically exact method for the analysis of spatially structured Markov processes. The method is based on a systematic perturbation expansion around the deterministic, non-spatial mean-field theory, using the theory of distributions to account for space and the underlying stochastic differential equations to account for stochasticity. As an example, we consider a spatial version of the Levins metapopulation model, in which the habitat patches are distributed in the d-dimensional landscape Rd in a random (but possibly correlated) manner. Assuming that the dispersal kernel is characterized by a length scale L, we examine how the behavior of the metapopulation deviates from the mean-field model for a finite but large L. For example, we show that the equilibrium fraction of occupied patches is given by p(0)+c/L(d)+O(L(-3d/2)), where p(0) is the equilibrium state of the Levins model and the constant c depends on p(0), the dispersal kernel, and the structure of the landscape. We show that patch occupancy can be increased or decreased by spatial structure, but is always decreased by stochasticity. Comparison with simulations show that the analytical results are not only asymptotically exact (as L-->infinity), but a good approximation also when L is relatively small.     

Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis

Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.     

Is biotic resistance enhanced by natural variation in diversity?

Theories linking diversity to ecosystem function have been challenged by the widespread observation of more exotic species in more diverse native communities. Few studies have addressed the underlying processes by dissecting how biotic resistance to new invaders may be shaped by the same environmental influences that determine diversity and other community properties. In grasslands with heterogeneous soils, we added invaders and removed competitors to analyze the causes of invasion resistance. Abiotic resistance was measured using invader success in the absence of the resident community. Biotic resistance was measured as the reduction in invader success in the presence of the resident community. Invaders were most successful where biotic resistance was lowest and abiotic resistance was highest, confirming the dominant role of biotic resistance. Contrary to theory, though, biotic resistance was highest where both species richness and functional diversity were lowest. In the multivariate framework of a structural equation model, biotic resistance was independent of community diversity, and was highest where fertile soils led to high community biomass. Seed predation slightly augmented biotic resistance without qualitatively changing the results. Soil‐related genotypic variation in the invader also did not affect the results. We conclude that in natural systems, diversity may be correlated with invasibility and yet have little effect on biotic resistance to invasion. More generally, the environmental causes of variation in diversity should be considered when examining the potential functional consequences of diversity.     

Using exclusion rate to unify niche and neutral perspectives on coexistence

The competitive exclusion principle is one of the most influential concepts in ecology. The classical formulation suggests a correlation between competitor species similarity and competition severity, leading to rapid competitive exclusion where species are very similar; yet neutral models show that identical species can persist in competition for long periods. Here, we resolve the conflict by examining two components of similarity – niche overlap and competitive similarity – and modeling the effects of each on exclusion rate (defined as the inverse of time to exclusion). Studying exclusion rate, rather than the traditional focus on binary outcomes (coexistence versus exclusion), allows us to examine classical niche and neutral perspectives using the same currency. High niche overlap speeds exclusion, but high similarity in competitive ability slows it. These predictions are confirmed by a well‐known model of two species competing for two resources. Under ecologically plausible scenarios of correlation between these two factors, the strongest exclusion rates may be among moderately similar species, while very similar and highly dissimilar competitors have very low exclusion rates. Adding even small amounts of demographic stochasticity to the model blurs the line between deterministic and probabilistic coexistence still further. Thus, focusing on exclusion rate, instead of on the binary outcome of coexistence versus exclusion, allows a variety of outcomes to result from competitive interactions. This approach may help explain species coexistence in diverse competitive communities and raises novel issues for future work.     

High-resolution sequence stratigraphy of a mixed carbonate-siliciclastic, cratonic ramp (Upper Ordovician; Kentucky–Ohio, USA): insights into the relative influence of eustasy and tectonics through analysis of facies gradients

Detailed facies analysis and event stratigraphy of an Upper Ordovician (Rocklandian–Edenian) cratonic ramp succession in eastern North America yields insights into eustatically driven sequence architecture and localized tectonic instability. Seven, predominantly subtidal, mixed carbonate-siliciclastic depositional sequences (3rd order) are identified and correlated across the length of a 275-km ramp–to–basin profile. Within the larger depositional sequences (3rd order) at least two smaller orders (4th and 5th) of cyclicity are recognizable. Three systems tracts occur within each sequence (transgressive, TST; highstand, HST; regressive, RST) and are considered in terms of their component parasequences (5th order). Generally, TSTs are composed of skeletal grainstone–rudstone facies, HSTs are dominated by shaly nodular wacke-packstone facies, and RSTs are mostly calcarenite facies. Systems tracts, sequence boundaries and their correlative conformities, maximum flooding surfaces, and forced regression surfaces were traced from shallow shelf to basinal settings. This high-resolution framework also provides insight into the timing of tectonic fluctuations on this cratonic ramp during the Taconic Orogeny and documents the relative influence of tectonism on lateral facies distributions and eustatically derived cyclicity.     

Comparative sequence stratigraphy of two classic Upper Ordovician successions, Trenton Shelf (New York–Ontario) and Lexington Platform (Kentucky–Ohio): implications for eustasy and local tectonism in eastern Laurentia

Comparison of the classic Upper Ordovician (Mohawkian to lower Cincinnatian; Caradoc to lower Ashgill) Black River and Trenton Groups in New York State/southern Ontario with the Tyrone–Lexington Formations exposed in the Jessamine Dome (southern Cincinnati Arch) in north-central Kentucky and southern Ohio reveals striking similarities. Previous emphasis on complex local facies mosaic has obscured widespread regional patterns. Biostratigraphy and K-bentonites provide broad constraints on inter-regional correlations; however, an allostratigraphic approach permits higher resolution correlations and a partial test of eustatic vs. strictly local tectonic models to explain stratigraphic patterns. Upper Mohawkian to lower Cincinnatian (455–449 Ma) depositional sequences, previously recognized for the Jessamine Dome and Nashville Dome areas, are correlated between the two main study areas and further refined; Chatfieldian (Rocklandian to Shermanian or Cobourgian of traditional terminology) sequences M5 and M6 of previous workers are interpreted to be composite sequences and are each subdivided into three smaller-scale sequences, which also have counterparts in the New York–Ontario strata. In turn, these correlations indicate at least partial allocyclic control on sedimentary cycles. Complex lateral variations within depositional sequences, especially in the late Shermanian to Edenian, indicate that tectonically controlled patterns of basinal subsidence and uplift of crustal blocks (perhaps reflecting forebulge migration) exerted a strong influence on the local facies and motif of depositional sequences. These tectonic features, however, did not obliterate the underlying allocyclic pattern. Indeed, high-resolution sequence stratigraphy enables detailed resolution of shifting patterns of minor uplift and subsidence.     

Blood viscosity in phocid seals: possible adaptations to diving

1. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of phocid seal red blood cells (RBC) are elevated compared to those of most terrestrial mammalian species. The influence of these characteristics on blood flow was revealed by viscosity (VIS) measurements. 2. RBC morphology and VIS of whole blood from 7 harbor seals and 5 northern elephant seals were compared with blood of the domestic pig. Samples were analysed for RBC count, white blood cell (WBC) count, total plasma proteins, hematocrit (HCT), MCV and MCHC. Viscosity measurements were made at shear rates from 11.5 to 230.4 s-1 on a Wells-Brookfield cone-plate viscometer at 37 degrees C. 3. Mean values for HCT (%), MCV (micron 3) and MCHC (%) were, respectively: elephant seal: 57, 176, 44; harbour seal: 53, 105, 38; domestic pig: 28, 54, 34. Pig blood was reconstituted to match seal blood HCTs. VIS determinations showed that seal and pig blood conform to the general mammalian dependence of VIS upon shear rate and HCT. 4. Seal blood VIS was 28% (harbour seal) and 16% (elephant seal) less than pig blood VIS at low shear (P less than 0.05). Seal blood carried more hemoglobin per unit volume than did pig blood reconstituted to the same HCT. Fewer, larger RBC with higher MCHC, and hence elevated oxygen storage, accompanied by reduced VIS and reduced flow resistance near stasis suggests that this feature of phocid seal blood is an adaptation to circulatory redistribution during long dives.