The Lysyl Oxidase Inhibitor, β-Aminopropionitrile,Diminishes the Metastatic Colonization Potential of Circulating Breast Cancer Cells

Lysyl oxidase (LOX), an extracellular matrix remodeling enzyme, appears to have a role in promoting breast cancer cell motility and invasiveness. In addition, increased LOX expression has been correlated with decreases in both metastases-free, and overall survival in breast cancer patients. With this background, we studied the ability of β-aminopropionitrile (BAPN), an irreversible inhibitor of LOX, to regulate the metastatic colonization potential of the human breast cancer cell line, MDA-MB-231. BAPN was administered daily to mice starting either 1 day prior, on the same day as, or 7 days after intracardiac injection of luciferase expressing MDA-MB-231-Luc2 cells. Development of metastases was monitored by in vivo bioluminescence imaging, and tumor-induced osteolysis was assessed by micro-computed tomography (μCT). We found that BAPN administration was able to reduce the frequency of metastases. Thus, when BAPN treatment was initiated the day before, or on the same day as the intra-cardiac injection of tumor cells, the number of metastases was decreased by 44%, and 27%, and whole-body photon emission rates (reflective of total tumor burden) were diminished by 78%, and 45%, respectively. In contrast, BAPN had no effect on the growth of established metastases. Our findings suggest that LOX activity is required during extravasation and/or initial tissue colonization by circulating MDA-MB-231 cells, lending support to the idea that LOX inhibition might be useful in metastasis prevention.     

Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin

Nearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold. Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasma-mass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.     

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation‐prone polyQ protein derived from human huntingtin. Expression of Q97‐GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97‐GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97‐GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post‐translational import of mitochondrial precursor proteins into mitochondria competes with aggregation‐prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate‐limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.     

Mdm36 Is a Mitochondrial Fission-promoting Protein in Saccharomyces cerevisiae

The division of mitochondrial membranes is a complex process mediated by the dynamin-related protein Dnm1 in yeast, acting in concert with several cofactors. We have identified Mdm36 as a mitochondria-associated protein required for efficient mitochondrial division. Δmdm36 mutants contain highly interconnected mitochondrial networks that strikingly resemble known fission mutants. Furthermore, mitochondrial fission induced by depolymerization of the actin cytoskeleton is blocked in Δmdm36 mutants, and the number of Dnm1 clusters on mitochondrial tips is reduced. Double mutant analyses indicate that Mdm36 acts antagonistically to fusion-promoting components, such as Fzo1 and Mdm30. The cell cortex-associated protein Num1 was shown previously to interact with Dnm1 and promote mitochondrial fission. We observed that mitochondria are highly motile and that their localization is not restricted to the cell periphery in Δmdm36 and Δnum1 mutants. Intriguingly, colocalization of Num1 and Dnm1 is abolished in the absence of Mdm36. These data suggest that Mdm36 is required for mitochondrial division by facilitating the formation of protein complexes containing Dnm1 and Num1 at the cell cortex. We propose a model that Mdm36-dependent formation of cell cortex anchors is required for the generation of tension on mitochondrial membranes to promote mitochondrial fission by Dnm1.     

Effects of developmental and functional interactions on mouse cranial variability through late ontogeny

The mammalian skull performs a variety of functions and its growth and development mirrors this complexity. Cranial growth and development have been actively studied for many years. Despite this interest, the variation in the patterns and processes of skull growth has attracted little attention. An important and unanswered question is the extent to which patterns of cranial covariation and variation are dynamically reworked throughout postnatal growth. To address this question, we examine patterns of variability in random-bred mouse skulls aged 35, 90, and 150 days. Using a battery of both Procrustes coordinate and Euclidean distance-based methods, we measure mean shape, canalization, developmental stability, and morphological integration in these skulls. We predict that the patterns of variability are dynamic, particularly between the youngest and the two oldest age groups due to the influence of functional effects such as postweaning mastication. We also hypothesize that patterns of variability are structured by the same functional and developmental factors that have been shown to influence cranial growth in primates. Our results indicate that contrary to our predictions, patterns of canalization, developmental stability, and morphological integration are stabilized before 35 days. The mean shape, however, changed significantly with growth. We found that only the facial region showed significant integration as predicted by the functional matrix model used in other studies of integration. These results indicate that phenotypic integration in these mice does not closely match those found for primate species, suggesting that comparisons between species should be made with care.     

The brachymorph mouse and the developmental-genetic basis for canalization and morphological integration

Although it is well known that many mutations influence phenotypic variability as well as the mean, the underlying mechanisms for variability effects are very poorly understood. The brachymorph (bm) phenotype results from an autosomal recessive mutation in the phosphoadenosine-phosphosulfate synthetase 2 gene (Papps2). A major cranial manifestation is a dramatic reduction in the growth of the chondrocranium which results from undersulfation of glycosaminoglycans (GAGs) in the cartilage matrix. We found that this reduction in the growth of the chondrocranium is associated with an altered pattern of craniofacial shape variation, a significant increase in phenotypic variance and a dramatic increase in morphological integration for craniofacial shape. Both effects are largest in the basicranium. The altered variation pattern indicates that the mutation produces developmental influences on shape that are not present in the wildtype. As the mutation dramatically reduces sulfation of GAGs, we infer that this influence is variation among individuals in the degree of sulfation, or variable expressivity of the mutation. This variation may be because of genetic variation at other loci that influence sulfation, environmental effects, or intrinsic effects. We infer that chondrocranial development exhibits greater sensitivity to variation in the sulfation of chondroitin sulfate when the degree of sulfation is low. At normal levels, sulfation probably contributes minimally to phenotypic variation. This case illustrates canalization in a particular developmental-genetic context.     

Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU,a prokaryotic homolog of the eukaryotic proteasome

In the prokaryotic homolog of the eukaryotic proteasome, HslUV, the "double donut" HslV protease is allosterically activated by HslU, an AAA protein of the Clp/Hsp100 family consisting of three (amino-terminal, carboxy-terminal, and intermediate) domains. The intermediate domains of HslU, which extend like tentacles from the hexameric ring formed by the amino-terminal and carboxy-terminal domains, have been deleted; an asymmetric HslU(DeltaI)(6)HslV(12) complex has been crystallized; and the structure has been solved to 2.5A resolution, revealing an assembly in which a HslU(DeltaI) hexamer binds one end of the HslV dodecamer. The conformation of the protomers of the HslU(DeltaI)-complexed HslV hexamer is similar to that in the symmetric wild-type HslUV complex, while the protomer conformation of the uncomplexed HslV hexamer is similar to that of HslV alone. Reaction in the crystals with a vinyl sulfone inhibitor reveals that the HslU(DeltaI)-complexed HslV hexamer is active, while the uncomplexed HslV hexamer is inactive. These results confirm that HslV can be activated by binding of a hexameric HslU(DeltaI)(6) ring lacking the I domains, that activation is effected through a conformational change in HslV rather than through alteration of the size of the entry channel into the protease catalytic cavity, and that the two HslV(6) rings in the protease dodecamer are activated independently rather than cooperatively.     

Mechanical,chemical and X-ray analysis of wood in the two tropical lianas<Emphasis Type="Italic"> Bauhinia guianensis</Emphasis> and<Emphasis Type="Italic"> Condylocarpon guianense</Emphasis>: variations during ontogeny

Mechanical and chemical properties as well as microfibril angles of wood tissues from different ontogenetic stages are determined for the neotropical lianas Bauhinia guianensis and Condylocarpon guianense. The mechanical properties include the elastic moduli under bending and under dynamic torsion. The chemical analyses cover (i) the content of cellulose, lignin and hemicelluloses fractions, (ii) the monomeric composition of the uncondensed lignin, and (iii) the composition of the hemicelluloses with respect to neutral monosaccharides. By comparing the wood properties of these lianas with the corresponding properties of wood from self-supporting deciduous trees, common characteristics and differences are revealed. Additionally, the changes in the lignin and polysaccharides fractions as well as the variations in microfibril orientation that occur during ontogeny of the two liana species are discussed with regard to their implications for the mechanical properties of wood.     

A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L

Apg8 is a ubiquitin-like protein involved in autophagy in yeast. Apg8 is covalently but transiently attached to membrane lipids through the actions of activating, conjugating, and processing/deconjugating enzymes. The mammalian Apg8 homologues GATE-16, GARARAP, and MAP1-LC3 have been implicated in intra-Golgi transport, receptor sorting, and autophagy, respectively. All are served by a single set of activating and conjugating enzymes. Here we identify a novel mammalian Apg8 homologue, which we name Apg8L, and describe the synthesis of electrophilic probes based on the GATE-16, GARARAP, MAP1-LC3, and Apg8L proteins. These probes not only form specific adducts in crude cell lysates, but also allow identification of the cellular proteases specific for the C termini of these Apg8 homologues. We find a single protease, Apg4B/autophagin-1, capable of acting on GATE-16, GABARAP, MAP1-LC3, and Apg8L. The Apg4B/autophagin-1 protease thus serves as a processing/deconjugating enzyme for these four highly divergent mammalian Apg8 homologues.