Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both β-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.     

Relative growth and morphological sexual maturity size of the freshwater crab Trichodactylus borellianus (Crustacea,Decapoda, Trichodactylidae) in the Middle Paraná River,Argentina

The relative growth of a number of morphological dimensions of the South American freshwater crab Trichodactylus borellianus (Trichodactylidae) were compared and related to sexual dimorphism. Crabs were collected from ponds in the Middle Paraná River in Argentina. A regression model with segmented relationship was used to test for relative growth between these measurements where breakpoints infer the body size at which crabs reach sexual maturity. In both sexes the carapace width and the length, height, and thickness of the right and left chelae were measured, as well as the male pleopod length and the female abdomen width. All of these measurements were found to show positive allometry with the exception of the male pleopod length and the left chelae, which did not show a breakpoint. In females the breakpoint for the abdomen width inferred a morphological sexual maturity at carapace width 6.9 mm. In males the break point for the pleopod length was at carapace width 6.6 mm, with that for the chelae measurements was between carapace widths 6.4 and 6.9 mm. The relative growth pattern in Trichodactylus borellianus was found to be similar to that recorded for other species of the family Trichodactylidae.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.     

The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs.     

MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway

Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.     

Ouabain-Induced Alterations of the Apical Junctional Complex Involve α1 and β1 Na,K-ATPase Downregulation and ERK1/2 Activation Independent of Caveolae in Colorectal Cancer Cells

Studies have reported that Na,K-ATPase interacts with E-cadherin to stabilize (AJs) and regulate the expression of claudins, the main proteins present in the tight junction (TJ) in epithelial cells containing caveolae. However, the role of this ATPase in the regulation of the AJ and TJ proteins in colorectal cancer cells as well as the molecular events underlying this event in a caveolae-independent system remain undefined. In the present study, we used ouabain, a classic drug known to inhibit Na,K-ATPase, and Caco-2 cells, which are a well-established human colorectal cancer model that does not exhibit caveolae. We demonstrated that ouabain treatment resulted in a reduction of the β1 Na,K-ATPase protein and cell redistribution of the AJ proteins E-cadherin and β-catenin, as well as the α1 Na,K-ATPase subunit. Furthermore, ouabain increased claudin-3 protein levels, impaired the TJ barrier function and increased cell viability and proliferation during the early stages of treatment. Additionally, the observed ouabain-induced events were dependent on the activation of ERK1/2 signaling; but in contrast to previous studies, this signaling cascade was caveolae-independent. In conclusion, our findings strongly suggest that α1 and β1 Na,K-ATPase downregulation and ERK1/2 activation induced by ouabain are interlinked events that play an important role during cell–cell adhesion loss, which is an important step during the tumor progression of colorectal carcinomas.     

The validity of ecogeographical rules is context‐dependent: testing for Bergmann's and Allen's rules by latitude and elevation in a widespread Andean duck

Consistent responses by various organisms to common environmental pressures represent strong evidence of natural selection driving geographical variation. According to Bergmann's and Allen's rules, animals from colder habitats are larger and have smaller limbs than those from warmer habitats to minimize heat loss. Although evidence supporting both rules in different organisms exists, most studies have considered only elevational or latitudinal temperature gradients. We tested for the effects of temperature associated with both elevation and latitude on body and appendage size of torrent ducks (Merganetta armata), a widespread species in Andean rivers. We found a negative relationship between body size and temperature across latitude consistent with Bergmann's rule, whereas there was a positive relationship between these variables along replicate elevational gradients at different latitudes. Limb‐size variation did not support Allen's rule along latitude, nor along elevation. High‐elevation ducks were smaller and had longer wings than those inhabiting lower elevations within a river. We hypothesize that temperature is likely a major selective pressure acting on morphology across latitudes, although hypoxia or air density may be more important along elevational gradients. We conclude that the effect of temperature on morphology, and hence the likelihood of documenting ecogeographical ‘rules’, depends on the environmental context in which temperature variation is examined. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 850–862.