Complex and dynamic expression of cadherins in the embryonic marmoset cerebral cortex

Cadherin is a cell adhesion molecule widely expressed in the nervous system. Previously, we analyzed the expression of nine classic cadherins (Cdh4, Cdh6, Cdh7, Cdh8, Cdh9, Cdh10, Cdh11, Cdh12, and Cdh20) and T‐cadherin (Cdh13) in the developing postnatal common marmoset (Callithrix jacchus) brain, and found differential expressions between mice and marmosets. In this study, to explore primate‐specific cadherin expression at the embryonic stage, we extensively analyzed the expression of these cadherins in the developing embryonic marmoset brain. Each cadherin showed differential spatial and temporal expression and exhibited temporally complicated expression. Furthermore, the expression of some cadherins differed from that in rodent brains, even at the embryonic stage. These results suggest the possibility that the differential expressions of diverse cadherins are involved in primate specific cortical development, from the prenatal to postnatal period.     

Two‐step d‐ononitol epimerization pathway in Medicago truncatula

Methylated inositol, d ‐pinitol (3‐O‐methyl‐d ‐chiro‐inositol), is a common constituent in legumes. It is synthesized from myo‐inositol in two reactions: the first reaction, catalyzed by myo‐inositol‐O‐methyltransferase (IMT), consists of a transfer of a methyl group from S‐adenosylmethionine to myo‐inositol with the formation of d ‐ononitol, while the second reaction, catalyzed by d ‐ononitol epimerase (OEP), involves epimerization of d ‐ononitol to d ‐pinitol. To identify the genes involved in d ‐pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co‐expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo‐keto reductase and MtOEPB, encoding a short‐chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo‐inositol to d ‐ononitol and showed that MtOEPA enzyme has NAD⁺‐dependent d ‐ononitol dehydrogenase activity, while MtOEPB enzyme has NADP⁺‐dependent d ‐pinitol dehydrogenase activity. Both enzymes are required for epimerization of d ‐ononitol to d ‐pinitol, which occurs in the presence of NAD⁺ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d ‐ononitol and d ‐pinitol in transformants. As this two‐step pathway of d ‐ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD⁺, we speculate that one of the functions of this pathway might be regeneration of NADP⁺ during drought stress.     

Pou‐V factor Oct25 regulates early morphogenesis in Xenopus laevis

POU‐V class proteins like Oct4 are crucial for keeping cells in an undifferentiated state. An Oct4 homologue in Xenopus laevis, Oct25, peaks in expression during early gastrulation, when many cells are still uncommitted. Nevertheless, extensive morphogenesis is taking place in all germ layers at that time. Phenotypical analysis of embryos with Oct25 overexpression revealed morphogenesis defects, beginning during early gastrulation and resulting in spina‐bifida‐like axial defects. Analysis of marker genes and different morphogenesis assays show inhibitory effects on convergence and extension and on mesoderm internalization. On a cellular level, cell–cell adhesion is reduced. On a molecular level, Oct25 overexpression activates expression of PAPC, a functional inhibitor of the cell adhesion molecule EP/C‐cadherin. Intriguingly, Oct25 effects on cell–cell adhesion can be restored by overexpression of EP/C‐cadherin or by inhibition of the PAPC function. Thus, Oct25 affects morphogenesis via activation of PAPC expression and subsequent functional inhibition of EP/C‐cadherin.     

Increasing l‐isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two‐component export system BrnFE

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.     

Methane emissions from six crop species exposed to three components of global climate change: temperature, ultraviolet-B radiation and water stress

The l ‐ascorbate (AsA) content and the expression of six l ‐galactose pathway‐related genes were analyzed in peach flesh during fruit development. Fluctuation of AsA during peach fruit development was divided into four phases based on the overall total AsA (T‐AsA) content per fruit: AsA I, 0–36 days after full bloom (DAFB); AsA II, 37–65 DAFB; AsA III, 66–92 DAFB and AsA IV, 93–112 DAFB. Phase AsA III was a lag phase for AsA accumulation, but did not coincide with the lag phase for fruit development. The T‐AsA concentration was highest at the early stage until 21 DAFB [2–3μmol per gram of fresh weight (g^?1 FW)], and decreased to 1/4 and 1/15 of this value at 50 and 92 DAFB, respectively. T‐AsA then remained at 0.15–0.20μmol g^?1 FW until harvest at 112 DAFB. More than 90% of the T‐AsA was in the reduced form until 21 DAFB. The proportion of reduced form of AsA then decreased concomitantly with the decrease in AsA concentration. To determine the main pathway of AsA biosynthesis and the AsA biosynthetic capacity of peach flesh, several precursors were incubated with immature whole fruit (59 DAFB). The AsA concentration increased markedly with l ‐galactono‐1,4‐lactone or l ‐galactose (Gal), but d ‐galacturonate and l ‐gulono‐1,4‐lactone failed to increase AsA, indicating dominance of the Gal pathway and potent AsA biosynthetic capabilities in immature peach flesh. The expression of genes involved in the last six steps of the Gal pathway was measured during fruit development. The genes studied included GDP‐d ‐mannose pyrophosphorylase (GMPH), GDP‐ d ‐mannose‐3′,5′‐epimerase (GME), GDP‐ l ‐galactose guanylyltransferase (GGGT), l ‐galactose‐1‐phosphate phosphatase (GPP), l ‐galactose‐1‐dehydrogenase (GDH) and l ‐galactono‐1,4‐lactone dehydrogenase (GLDH). GMPH, GME and GGGT had similar expression patterns that peaked at 43 DAFB. GPP, GDH and GLDH also had similar expression patterns that peaked twice at 21 and 91 DAFB, although the expression of GDH was quite low. High level of T‐AsA concentration was roughly correlated with the level of gene expression in the early period of fruit development (AsA I), whereas no such relationships were apparent in the other periods (e.g. AsA III and IV). On the basis of these findings, we discuss the regulation of AsA biosynthesis in peach fruit.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

Molecular mechanism underlying pseudopeloria in Habenaria radiata (Orchidaceae)

Habenaria radiata (Orchidaceae) has two whorls of perianth, comprising three greenish sepals, two white petals and one lip (labellum). By contrast, the pseudopeloric (with a decreased degree of zygomorphy) mutant cultivar of H. radiata , ‘Hishou’, has changes in the identities of the dorsal sepal to a petaloid organ and the two ventral sepals to lip‐like organs. Here, we isolated four DEFICIENS ‐ like and two AGL 6 ‐like genes from H. radiata , and characterized their expression. Most of these genes revealed similar expression patterns in the wild type and in the ‘Hishou’ cultivar, except Hr DEF ‐C3. The Hr DEF ‐C3 gene was expressed in petals and lip in the wild type but was ectopically expressed in sepal, petals, lip, leaf, root and bulb in ‘Hishou’. Sequence analysis of the Hr DEF ‐C3 loci revealed that the ‘Hishou’ genome harbored two types of Hr DEF ‐C3 genes: one identical to wild‐type Hr DEF ‐C3 and the other carrying a retrotransposon insertion in its promoter. Genetic linkage analysis of the progeny derived from an intraspecific cross between ‘Hishou’ and the wild type demonstrated that the mutant pseudopeloric trait was dominantly inherited and was linked to the Hr DEF ‐C3 gene carrying the retrotransposon. These results indicate that the pseudopeloric phenotype is caused by retrotransposon insertion in the Hr DEF ‐C3 promoter, resulting in the ectopic expression of Hr DEF ‐C3 . As the expression of Hr AGL 6‐C2 was limited to lateral sepals and lip, the overlapping expression of Hr DEF ‐C3 and Hr AGL 6‐C2 is likely to be responsible for the sepal to lip‐like identity in the lateral sepals of the ‘Hishou’ cultivar.     

Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type‐2 and Their Major Compounds

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non‐cytotoxic and anti‐dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP‐SPE method allowed isolating the antiviral flavanone (2S)‐isosakuranetin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 1 ) from both species and yielded less active sub‐fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3′,5′‐trihydroxyflavanone‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 2a / 2b ) from F. hyacinthina; the known narigenin‐7‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 3 ) from both species; rutin ( 4 ) and quercetin‐4′‐β‐d ‐O‐glucopyranosyl‐3‐O‐rutinoside ( 5 ) from F. hyacinthina, and kaempferol‐3‐O‐rutinoside ( 6 ), erythroxyloside A ( 7 ) and asperuloside ( 8 ) from F. truncata have been isolated from these sub‐fractions. Compounds 4  –  8 are reported for the first time in Faramea spp.     

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen–host interactions

Enteric pathogen–host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane‐bound toll‐like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro‐inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase‐1 and are regulated by related caspases, such as caspase‐11, ‐4, ‐5 and ‐8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome‐mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

Transplantation of photoreceptor precursor cells into the retina of an adult Drosophila

Blindness caused by the disconnection between photoreceptor cells and the brain can be cured by restoring this connection through the transplantation of retinal precursor neurons. However, even after transplanting these cells, it is still unclear how to guide the axons over the long distance from the retina to the brain. To establish a method of guiding the axons of transplanted neurons, we used the Drosophila visual system. By testing different conditions, including the dissociation and preincubation length, we have successfully established a method to transplant photoreceptor precursor cells isolated from the developing eye discs of third‐instar larvae into the adult retina. Moreover, we overexpressed N‐cadherin (CadN) in the transplant, since it is known to be broadly expressed in the optic lobe well after developmental stages, continuing through adult stages. We found that promoting the cell adhesive properties using CadN enhances the axonal length of the grafted photoreceptor neurons and therefore is useful for future transplantation. We tested the overexpression of a CadN::Frazzled chimeric receptor and found that there was no difference in axonal length from our wild‐type transplants, suggesting that the intracellular domain of CadN is necessary for axonal elongation. Altogether, using the Drosophila visual system, we have established an excellent platform for exploring the molecules required for proper axon extension of transplanted neuronal cells. Future studies building from this platform will be useful for regenerative therapy of the human nervous system based on transplantation.     

Comparative analysis of the reactive oxygen species‐producing enzymatic activity of Arabidopsis NADPH oxidases

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, ‐D, ‐F, ‐H and ‐J) are synergistically activated by Ca²⁺‐binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS‐producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, ‐H and ‐J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or ‐J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.     

A proteomic approach to understand MMP‐3‐driven developmental processes in the postnatal cerebellum: Chaperonin CCT6A and MAP kinase as contributing factors

Matrix metalloproteinase‐3 (MMP‐3) deficiency in mice was previously reported to result in a transiently retarded granule cell migration at postnatal day 8 (P8) and a sustained disturbed arborization of Purkinje cell dendrites from P8 on, concomitant with a delayed synapse formation between granule cells and Purkinje cells and resulting in mild deficits in motor performance in adult animals. However, the molecular mechanisms by which MMP‐3 contributes to proper development of the cerebellar cortex during the first postnatal weeks remains unknown. In this study, we used a functional proteomics approach to investigate alterations in protein expression in postnatal cerebella of wild‐type versus MMP‐3 deficient mice, and to further elucidate MMP‐3‐dependent pathways and downstream targets in vivo. At P8, two‐dimensional difference gel electrophoresis and mass spectrometry identified 20 unique proteins with a different expression between the two genotypes. Subsequent “Ingenuity Pathway Analysis” and Western blotting indicate that the chaperonin containing T‐complex polypeptide 1, subunit 6A and the MAP kinase signaling pathway play a key role in the MMP‐3‐dependent regulation of neurite outgrowth and neuronal migration in the developing brain. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1033–1048, 2015     

Expression of tomato reference genes using established primer sets: Stability across experimental set‐ups

Reliable reference genes are critical for relative quantification using quantitative real‐time PCR (qPCR). Ten tomato genes (Solanum lycopersicum) and their respective primer sets, which have been used over the last 6 years as references in expression studies, were evaluated for their performance using leaf tissue samples grown under semi‐controlled conditions and infected with grey mould (Botrytis cinerea) or late blight (Phytophthora infestans). The target genes coding for U6 snRNA‐associated Sm‐like protein LSm7, calcineurin B‐like protein and V‐type proton ATPase were the most stable expressed of all the genes tested in three experimental repetitions. Evaluation of candidate reference genes with geNorm and NormFinder softwares yielded the lowest mean values for their respective primer sets LSM7, SlCBL1 and SlATPase, suggesting stable expression. However, SlATPase primer set revealed a comparably high intra‐group variation and was thus not considered further. In follow‐up experiments with P. infestans, the geNorm and NormFinder values of primer sets LSM7 and SlCBL1 were even lower, indicating the stability of their expression also under these conditions. Primer efficiency differed by ‐18 to +5 percentage points from values presented in the literature. Our findings show that a reference primer set which delivers the best results in one system may be outperformed by another under different experimental conditions, thus recommending a reassessment of both expression stability and qPCR efficiency whenever the biological or technical experimental set‐up is changed. On the basis of our results, we recommend the use of LSM7 and SlCBL1 as reference primer sets for gene expression studies on plant tissue derived from open or semi‐controlled conditions.