High synteny and colinearity among Eucalyptus genomes revealed by high-density comparative genetic mapping

Understanding genome differentiation is important to compare and transfer genomic information between taxa, such as from model to non-model organisms. Comparative genetic mapping can be used to assess genome differentiation by identifying similarities and differences in chromosome organization. Following release of the assembled Eucalyptus grandis genome sequence (January 2011; ), a better understanding of genome differentiation between E. grandis and other commercially important species belonging to the subgenus Symphyomyrtus is required. In this study, comparative genetic mapping analyses were conducted between E. grandis, Eucalyptus urophylla, and Eucalyptus globulus using high-density linkage maps constructed from Diversity Array Technology and microsatellite molecular markers. There were 236–393 common markers between maps, providing the highest resolution yet achieved for comparative mapping in Eucalyptus. In two intra-section comparisons (section Maidenaria–E. globulus and section Latoangulatae–E. grandis vs. E. urophylla), ∼1% of common markers were non-syntenic and within chromosomes 4.7–6.8% of markers were non-colinear. Consistent with increasing taxonomic distance, lower synteny (6.6% non-syntenic markers) was observed in an inter-section comparison between E. globulus and E. grandis × E. urophylla consensus linkage maps. Two small chromosomal translocations or duplications were identified in this comparison representing possible genomic differences between E. globulus and section Latoangulatae species. Despite these differences, the overall high level of synteny and colinearity observed between section Maidenaria–Latoangulatae suggests that the genomes of these species are highly conserved indicating that sequence information from the E. grandis genome will be highly transferable to related Symphyomyrtus species.     

Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

Pyridoxamine protects proteins from functional damage by 3-deoxyglucosone: mechanism of action of pyridoxamine

Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell-matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952-2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyglucosone (3-DG) can damage protein functionality, including the ability of collagen IV to interact with glomerular mesangial cells. We have also demonstrated that PM can protect against 3-DG-induced protein damage via a novel mechanism that includes transient adduction of 3-DG by PM followed by irreversible PM-mediated oxidative cleavage of 3-DG. Our results suggest that, in diabetic nephropathy, the therapeutic effect of PM is achieved, in part, via protection of renal cell-matrix interactions from damage by a variety of RCS. Our data emphasize the potential importance of the contribution by 3-DG, along with other more reactive RCS, to this pathogenic mechanism.     

Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.     

Goodpasture antigen-binding protein and its spliced variant, ceramide transfer protein, have different functions in the modulation of apoptosis during zebrafish development

Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.     

Identification of noncollagenous sites encoding specific interactions and quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen: implications for Alport gene therapy

Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.     

Biological and molecular characteristics of Beauveria bassiana isolates from California Lygus hesperus (Hemiptera: Miridae) populations

Lygus hesperus is an important pest of many crops grown in the Western US. In addition, other species of Lygus cause damage in other parts of the world. To date, no selective pesticide exists for the control of Lygus spp. and broad spectrum pesticides that also kill natural enemies may lead to secondary pests. Entomopathogenic fungi may offer an alternative to chemical pesticides. Isolates of Beauveria bassiana collected from San Joaquin Valley of California (SJV) L. hesperus populations were screened for their ability to grow at high temperatures and for their ability to infect and kill L. hesperus adults and nymphs under laboratory conditions. No isolate grew at 37 or 35 °C but most isolates were able to grow at 32 °C. In addition, one L. hesperus isolate was more efficacious at higher doses than the commercial isolate. Microsatellite markers were used to determine that selected isolates could be distinguished from other isolates. Preliminary information suggested 82 SJV isolates of B. bassiana were closely related to each other but distantly related to the commercial isolate.     

A new series of neutral 5-substituted 4-anilinoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase

Starting from initial lead 1 containing a basic 5-substituent, optimisation of the glycolamide-derived neutral 5-substituent led to potent inhibitors of erbB2 with good pharmacokinetics. Representative compounds 19 and 21 inhibited phosphorylation of erbB2 in a mouse BT474C xenograft model after oral administration.