Cripto binds transforming growth factor beta (TGF-beta) and inhibits TGF-beta signaling

Cripto is a developmental oncoprotein and a member of the epidermal growth factor-Cripto, FRL-1, Cryptic family of extracellular signaling molecules. In addition to having essential functions during embryogenesis, Cripto is highly expressed in tumors and promotes tumorigenesis. During development, Cripto acts as an obligate coreceptor for transforming growth factor beta (TGF-beta) ligands, including nodals, growth and differentiation factor 1 (GDF1), and GDF3. As an oncogene, Cripto is thought to promote tumor growth via mechanisms including activation of mitogenic signaling pathways and antagonism of activin signaling. Here, we provide evidence supporting a novel mechanism in which Cripto inhibits the tumor suppressor function of TGF-beta. Cripto bound TGF-beta and reduced the association of TGF-beta with its type I receptor, TbetaRI. Consistent with its ability to block receptor assembly, Cripto suppressed TGF-beta signaling in multiple cell types and diminished the cytostatic effects of TGF-beta in mammary epithelial cells. Furthermore, targeted disruption of Cripto expression by use of small inhibitory RNA enhanced TGF-beta signaling, indicating that endogenous Cripto plays a role in restraining TGF-beta responses.     

Coevolution Predicts Direct Interactions between mtDNA-Encoded and nDNA-Encoded Subunits of Oxidative Phosphorylation Complex I

Despite years of research, the structure of the largest mammalian oxidative phosphorylation (OXPHOS) complex, NADH-ubiquinone oxidoreductase (complex I), and the interactions among its 45 subunits are not fully understood. Since complex I harbors subunits encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genomes, with the former evolving ∼ 10 times faster than the latter, tight cytonuclear coevolution is expected and observed. Recently, we identified three nDNA-encoded complex I subunits that underwent accelerated amino acid replacement, suggesting their adjustment to the elevated mtDNA rate of change. Hence, they constitute excellent candidates for binding mtDNA-encoded subunits.Here, we further disentangle the network of physical cytonuclear interactions within complex I by analyzing subunits coevolution. Firstly, relying on the bioinformatic analysis of 10 protein complexes possessing solved structures, we show that signals of coevolution identified physically interacting subunits with nearly 90% accuracy, thus lending support to our approach. When applying this approach to cytonuclear interaction within complex I, we predict that the ‘rate-accelerated’ nDNA-encoded subunits of complex I, NDUFC2 and NDUFA1, likely interact with the mtDNA-encoded subunits ND5/ND4 and ND5/ND4/ND1, respectively. Furthermore, we predicted interactions among mtDNA-encoded complex I subunits. Using the yeast two-hybrid system, we experimentally confirmed the predicted interactions of human NDUFC2 with ND4, the interactions of human NDUFA1 with ND1 and ND4, and the lack of interaction of NDUFC2 with ND3 and NDUFA1, thus providing a proof of concept for our approach.Our study shows, for the first time, evidence for direct interactions between nDNA-encoded and mtDNA-encoded subunits of human OXPHOS complex I and paves the path towards deciphering subunit interactions within complexes lacking three-dimensional structures. Our subunit-interactions-predicting method, ComplexCorr, is available at http://webclu.bio.wzw.tum.de/complexcorr.     

Growth modulation of transgenic potato plants by heterologous expression of bacterial carbohydrate-binding module

Transgenic potato plants (Solanum tuberosum cv. Desiree) expressing the bacterial carbohydrate-binding module (CBM) family III, which is part of the Clostridium cellulovorans CBPA, under control of the CaMV 35S promoter were employed to investigate the influence of this protein on plant development. Eleven independent transgenic plants were found to express the cbm gene, at levels varying from one to four copies. Relative to non-transgenic controls, CBM-expressing plants were characterized by significantly more rapid elongation of the main stem. In addition, under both greenhouse and field conditions, the emergence rate of these plants was higher than in the controls, and their leaf area at early stages of development was larger, resulting in faster accumulation of fresh and dry weight than in control plants. Determination of cell size indicated that epidermal cells in young tissue were significantly larger in CBM-expressing than in control potato plants. These findings suggest that the CBM influence at the cellular level my cause significant alterations in plant growth both in tissue culture and in vivo under field conditions.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.     

Abnormal `wrinkled' cell walls and retarded development of transgenic Arabidopsis thaliana plants expressing endo-1,4-β-glucanase (cell) antisense

Transgenic Arabidopsis thaliana plants expressing cell antisense exhibit reduced levels of cell mRNA and protein compared with wild-type plants. The former display significant alterations in their phenotype. cell antisense plants have shorter stems and roots and are mechanically weaker than their wild-type counterparts. In cell antisense plants, the cell wall structure is markedly disrupted: both fluorescent confocal microscopy and scanning electron microscopy revealed `wrinkled' cell walls, thus indicating that CEL1 plays an important role in cell wall relaxation during cell growth and expansion. In cell antisense plants, the number of xylem elements per bundle is smaller than in the wild-type. In addition, both xylem elements and interfascicular fibers are significantly less lignified in the former. It is suggested that in A. thaliana, abnormal cell wall deposition affected by CEL1 depletion is associated not only with cell growth, but also with the differentiation process in the vascular and supporting tissues.Equal contributors     

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.     

Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic significance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte deficiency, prenatal lethality has so far precluded analysis of postnatal consequences in the retina. We now report that endothelium-restricted ablation of platelet-derived growth factor-B generates viable mice with extensive inter- and intra-individual variation in the density of pericytes throughout the CNS. We found a strong inverse correlation between pericyte density and the formation of a range of retinal microvascular abnormalities strongly reminiscent of those seen in diabetic humans. Proliferative retinopathy invariably developed when pericyte density was <50% of normal. Our data suggest that a reduction of the pericyte density is sufficient to cause retinopathy in mice, implying that pericyte loss may also be a causal pathogenic event in human diabetic retinopathy.     

Sibling species of bean bruchids: a morphological and phylogenetic study of Acanthoscelides obtectus Say and Acanthoscelides obvelatus Bridwell

Acanthoscelides Schilsky is a large genus of neotropical bruchid beetles, in which most species show host plant specialization. Acanthoscelides obtectus and Acanthoscelides obvelatus are two sibling species specialized on Phaseolus beans, and are therefore considered pests. Up to now, the status of these two taxa has remained unclear, the few studies conducted having failed to elucidate whether these are two differentiated species or a single morphologically variable species. In addition, A. obvelatus has not been taken into account in the great majority of studies of bean bruchids. In this morphological and genetic study, we show that A. obtectus and A. obvelatus are two 'true' non-hybridizing species, which diverged about 22 Mya. Although the two species demonstrate only few morphological differences, we point out some diagnostic characters that enable their identification in the field. We also address a genetic method of differentiation of the two species, based on species-specific microsatellite loci. The strong morphological resemblance of these two species, despite their ancient divergence, may be the result of evolutionary stasis, which could be the consequence of stabilizing selection. Niche differentiation could enable the two species to coexist indefinitely.     

Specific combinations of human serum albumin introns direct high level expression of albumin in transfected COS cells and in the milk of transgenic mice

A new series of expression vectors, each comprised of the -lactoglobulin (BLG) promoter driving one of a variety of human serum albumin (HSA) minigenes or the entire gene, were evaluated for their ability to direct expression of HSAin vitro in COS tissue culture cells and into the milk of transgenic mice. Vectors directed a hierarchy of expression levelsin vitro, dependent upon the specific complement of HSA introns included. HSA introns acted in a synergistic manner. In addition, minigenes comprised of specific subsets of introns were more efficacious than the entire HSA gene with all of its introns. Transgenic mice expressed as much as 10 mg ml^–1 of HSA in their milk. Vectors comprised of specific intron subsets directed levels at 1 mg ml^–1 or greater in the milk of 20% of generated transgenics. A statistical correlation between the expression level trendin vitro with the trend of expressionin vivo (% which express) at detectable levels (p=0.0015) and at the level of greater than 0.1 mg ml^–1 (p=0.0156) was demonstrated. A weak correlation existed (p=0.0526) atin vivo levels of 1 mg ml^–1 or greater. These new vectors are expected to direct the production of high levels of HSA in the milk of a large percentage of generated transgenic dairy animals.