Integrin-mediated regulation of neurovascular development,physiology and disease

The mammalian central nervous system (CNS) is comprised of billions of neurons and glia that are intertwined with an elaborate network of blood vessels. These various neural and vascular cell types actively converse with one another to form integrated, multifunctional complexes, termed neurovascular units. Cell-cell communication within neurovascular units promotes normal CNS development and homeostasis, and abnormal regulation of these events leads to a variety of debilitating CNS diseases. This review will summarize (1) cellular and molecular mechanisms that regulate physiological assembly and maintenance of neurovascular units; and (2) signaling events that induce pathological alterations in neurovascular unit formation and function. An emphasis will be placed on neural-vascular cell adhesion events mediated by integrins and their extracellular matrix (ECM) ligands. I will highlight the role of a specific adhesion and signaling axis involving αvβ8 integrin, latent transforming growth factor β''s (TGFβ''s), and canonical TGFβ receptors. Possible functional links between components of this axis and other signal transduction cascades implicated in neurovascular development and disease will be discussed. Comprehensively understanding the pathways that regulate bidirectional neural-vascular cell contact and communication will provide new insights into the mechanisms of neurovascular unit development, physiology and disease.Key words: αvβ8 integrin, latent TGFβ, neurovascular unit, brain angiogenesis, cerebral hemorrhage     

The maize viviparous15 locus encodes the molybdopterin synthase small subunit

A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15-specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5' untranslated region as well as a micro-synteny among the cereals.     

A cationic chalcogenoxanthylium photosensitizer effective in vitro in chemosensitive and multidrug-resistant cells

Pentacyclic thio- (1) and seleno- (2) analogues of tetramethylrosamine (TMR) were prepared with a julolidyl fragment replacing the 3-dimethylamino substituent in the xanthylium core. The pentacylic structure increases the lipophilicity of 1 and 2 relative to TMR-S and TMR-Se and locks the lone-pair of electrons on the julolidyl N atom into conjugation with the xanthylium core. This conformational rigidization leads to longer wavelengths of absorption, but has little impact on other photophysical properties such as quantum yields for fluorescence and singlet-oxygen generation and fluorescence lifetimes in 1 and 2 relative to TMR-S and TMR-Se. Both 1 and 2 are effective photosensitizers against chemosensitive AUXB1 cells in vitro at 1 × 10⁻⁷ M and compound 2 is an effective photosensitizer against multidrug-resistant CR1R12 cells in vitro at 1 × 10⁻⁷ M. While the uptake TMR-S into CR1R12 cells as measured by fluorescence is significantly lower than uptake into chemosensitive AUXB1 cells, there is no significant difference in the uptake of 1 into either AUXB1 or CR1R12 cells. The addition of 2 × 10⁻⁴ M verapamil to the cells prior to treatment with 1 had no significant effect on the uptake of 1 into either AUXB1 or CR1R12 cells. Treating lipid-activated, purified Pgp with 2 and light gave complete inhibition of Pgp ATPase activity.     

Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.     

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in <Emphasis Type="Italic">Gossypium barbadense</Emphasis> L. accession GB713

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P₁, P₂, F₁, F₂, BC₁P₁, and BC₁P₂ generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F₂ population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren ^barb1 and Ren ^barb2 to designate these QTLs on chromosome 21 and Ren ^barb3 on chromosome 18.     

Comparisons of four approximation algorithms for large-scale linkage map construction

Efficient construction of large-scale linkage maps is highly desired in current gene mapping projects. To evaluate the performance of available approaches in the literature, four published methods, the insertion (IN), seriation (SER), neighbor mapping (NM), and unidirectional growth (UG) were compared on the basis of simulated F₂ data with various population sizes, interferences, missing genotype rates, and mis-genotyping rates. Simulation results showed that the IN method outperformed, or at least was comparable to, the other three methods. These algorithms were also applied to a real data set and results showed that the linkage order obtained by the IN algorithm was superior to the other methods. Thus, this study suggests that the IN method should be used when constructing large-scale linkage maps.     

Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.)

Acetolactate synthase (ALS) is responsible for a rate-limiting step in the synthesis of essential branched-chain amino acids. Resistance to ALS-inhibiting herbicides, such as trifloxysulfuron sodium (Envoke^®), can be due to mutations in the target gene itself. Alternatively, plants may exhibit herbicide tolerance through reduced uptake and translocation or increased metabolism of the herbicide. The diverse family of cytochrome P450 proteins has been suggested to be a source of novel herbicide metabolism in both weed and crop plants. In this study we generated a mapping population between resistant and susceptible cotton (Gossypium hirsutum L.) cultivars. We found that both cultivars possess identical and sensitive ALS sequences; however, the segregation of resistance in the F2 progeny was consistent with a single dominant gene. Here we report the closely linked genetic markers and approximate physical location on chromosome 20 of the source of Envoke herbicide susceptibility in the cotton cultivar Paymaster HS26. There are no P450 proteins in the corresponding region of the G. raimondii Ulbr. genome, suggesting that an uncharacterized molecular mechanism is responsible for Envoke herbicide tolerance in G. hirsutum. Identification of this genetic mechanism will provide new opportunities for exploiting sulfonylurea herbicides for management of both weeds and crop plants.