Modulations in gene expression and mapping of genes associated with cyst nematode infection of soybean

Infection of the soybean root by the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) induces a well-documented, yet poorly understood, response by the host plant. The plant response, involving the differentiation of a feeding structure, or "syncytium," facilitates the feeding and reproduction of the nematode to the detriment of the host. We used a genetic system involving a single dominant soybean gene conferring susceptibility to an inbred nematode strain, VL1, to characterize the nematode-host interaction in susceptible line PI 89008. The restriction fragment length polymorphism marker pB053, shown to map to a major SCN resistance locus, cosegregates with resistance among F2 progeny from the PI 89008 x PI 88287 cross. Cytological examination of the infection process confirmed that syncytium development in this genetic system is similar to that reported by others who used noninbred nematode lines. Our study of infected root tissue in the susceptible line PI 89008 revealed a number of genes enhanced in expression. Among these are catalase, cyclin, elongation factor 1alpha, beta-1,3-endoglucanase, hydroxy-methylglutaryl coenzyme A reductase, heat shock protein 70, late embryonic abundant protein 14, and formylglycinamidine ribonucleotide synthase, all of which we have genetically positioned on the public linkage map of soybean. Formylglycinamidine ribonucleotide synthase was found to be tightly linked with a major quantitative trait locus for SCN resistance. Our observations are consistent with the hypothesis proposed by others that feeding site development involves the dramatic modulation of gene expression relative to surrounding root cells.     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Adult habitat preferences, larval dispersal, and the comparative phylogeography of three Atlantic surgeonfishes (Teleostei: Acanthuridae)

Although many reef fishes of the tropical Atlantic are widely distributed, there are large discontinuities that may strongly influence phylogeographical patterns. The freshwater outflow of the Amazon basin is recognized as a major barrier that produces a break between Brazilian and Caribbean faunas. The vast oceanic distances between Brazil and the mid-Atlantic ridge islands represent another formidable barrier. To assess the relative importance of these barriers, we compared a fragment of the mitochondrial DNA (mtDNA) cytochrome b gene among populations of three species of Atlantic surgeonfishes: Acanthurus bahianus, A. chirurgus and A. coeruleus. These species have similar life histories but different adult habitat preferences. The mtDNA data show no population structure between Brazil and the mid-Atlantic islands, indicating that this oceanic barrier is readily traversed by the pelagic larval stage of all three surgeonfishes, which spend approximately 45-70 days in the pelagic environment. The Amazon is a strong barrier to dispersal of A. bahianus (d = 0.024, phiST = 0.724), a modest barrier for A. coeruleus (phiST = 0.356), and has no discernible effect as a barrier for A. chirurgus. The later species has been collected on soft bottoms with sponge habitats under the Amazon outflow, indicating that relaxed adult habitat requirements enable it to readily cross that barrier. A limited ability to use soft bottom habitats may also explain the low (but significant) population structure in A. coeruleus. In contrast, A. bahianus has not been collected over deep sponge bottoms, and rarely settles outside shallow reefs. Overall, adult habitat preferences seem to be the factor that differentiates phylogeographical patterns in these reef-associated species.     

Partial characterization of a tapeworm-secreted signal factor inducing sustained spike potentials in the smooth muscle of the rat small intestine

The rat tapeworm Hymenolepis diminuta alters the myoelectric activity of the small intestine. To determine if secreted factors from the tapeworm are responsible for these alterations of intestinal smooth muscle activity, tapeworm-conditioned medium (TCM) obtained from in vitro culture was infused via an indwelling cannula into the duodenum of an uninfected rat. Myoelectric recordings were analyzed for sustained spike potentials (SSP) and repetitive bursts of action potentials (RBAP), the previously characterized tapeworm modifications of the normal interdigestive myoelectric pattern. Results indicated that TCM initiated SSP, but not RBAP in the intestine of the uninfected rat. The SSP-inducing signal factor activity, present in TCM, was retained after boiling, prolonged freezing, proteinase treatment, and passage through a 10-kDa exclusion filter. The signal factor was soluble in the aqueous phase on lipid extraction. It was concluded that the SSP-inducing signal factor is a nonproteinaceous, heat-resistant, low-molecular weight, water soluble molecule.     

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.     

Cytochrome b562 variants: a library for examining redox potential evolution

A general understanding of how cytochromes evolve within a fixed structure to optimize redox potential for specific bioenergetic processes does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential which occurs when all sequence space available through mutation at two positions is examined within a fixed structural motif. Random mutation of Phe61 and Phe65 of cytochrome b562 (E. coli), and subsequent examination of a statistically significant sampling of this library, demonstrates that the redox potential can vary over 100 mV (>25% of the known accessible potential in native proteins with axial His-Met ligation) through mutation at these two positions. The redox potential of the wild-type protein occurs at an extremum of the distribution observed, indicating that Phe61 and Phe65 were most likely naturally selected to differentially stabilize the reduced state of the protein. At the other extremum, a compositionally conservative set of mutations (F61I, F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. NMR analyses indicate that a charge-dipole interaction which results from mutation of phenylalanine to tyrosine at position 65 may be responsible.     

In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site

The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.     

Serum immunoglobulin type G concentrations in calves produced by IVF and delivered by elective cesarean section

Colostrum ingestion by neonatal calves is widely recognized to provide passive transfer of immunity. In this study immunoglobulin absorption from colostrum was evaluated in 54 IVF-produced calves. The IVF calves were delivered by Cesarean section on Days 275 to 277 of gestation, 24 h after the dams had been administered 30 mg dexamethasone. The calves suckled bottles or were force-fed 6 L of colostrum in the first 12 h of life. Colostrum was obtained from the first post-calving milking of recipient dams or from frozen storage reserves if dam secretion was not adequate. Immunoglobulin type G (IgG) content of both sources of colostrum was determined. Serum samples from the calves were collected at 0, 12 and 24 h of age and analyzed for IgG. Twenty dairy calves born vaginally served as the controls and were subjected to the same colostrum management protocol except that the colostrum was obtained only from frozen post-calving milk of dairy cows from the same farm. The control calves were also subjected to the same sampling protocol. The IVF group of calves ingested more IgG (P < 0.0001) and absorbed more IgG by 24 h of age (P < 0.0001) than their control group counterparts. Absorption of IgG was analyzed by comparing the g/kg body weight of IgG with serum IgG values at corresponding times after birth. Colostrum absorption efficiency was the same for both IVF and control groups of calves at 12 and 24 h of age. There was a maximum IgG dose above which additional increases in serum IgG were not realized. The slightly premature, Cesarean delivered IVF calves absorbed IgG from colostrum similarly to control calves delivered vaginally.     

Molecular Phylogeny and Biogeography of the Marine ShrimpPenaeus

The evolutionary relationships among 13 species representing all six subgenera of the shrimp genusPenaeuswere examined using 558 bp of mitochondrial (mt) DNA from the cytochrome oxidase subunit I gene. Analyses of this sequence revealed high genetic divergence between species (d = 8–24%), a finding which contrasts with previous work, which indicated that genetic diversity, based on electrophoretic analysis of allozymes, was extremely low inPenaeus.Three tree-building methods (maximum parsimony, neighbor joining, and maximum likelihood) were concordant in indicating that current subgenera assignments do not reflect evolutionary partitions within the genusPenaeus.While the molecular phylogenies cast doubt on the validity of subgenera, the observed relationships are concordant with biogeographic boundaries across the tropical range ofPenaeus.Both the western Atlantic and eastern Pacific contain monophyletic species pairs which cluster together in all analyses. The Indo-Pacific contains a putative basal taxa (P. indicus), the deepest mtDNA lineages, and the highest diversity, including representatives of all three primary lineages observed inPenaeus.These data are consistent with the suggestion by Dallet al.(1990) thatPenaeusarose in the Indo-Pacific and radiated eastward and westward to account for the current circumtropical distribution of the genus. This phylogenetic framework forPenaeuswill enhance the scientific foundations for wildlife resource management and breeding experiments (hybridization and related manipulations) designed to improve the commercial value of captive strains.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.