Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.     

Functional and genetic survey of all known single-nucleotide polymorphisms within the human deoxyribonuclease I gene in wide-ranging ethnic groups

The single-nucleotide polymorphisms (SNPs) in the human DNase I gene (DNASE1) might be involved in susceptibility to some common diseases; however, only limited population data are available. Further, the effects of these SNPs on in vivo DNase I activity remain unknown. The genotype and haplotype of all the SNPs in DNASE1 were determined in 3 ethnic groups including 14 populations using newly developed methods. Together with our previous data on the nonsynonymous SNPs, two major haplotypes based on the five exonic SNPs were identified; genetic diversity in the Asian population was low. Among 10 SNPs, other than exonic SNPs in the gene, only 3 were polymorphic among all the populations. Haplotype distribution, based on all the polymorphic SNPs, was clarified to be generally varied in an ethnic-dependent manner. Thus, the genetic aspects of DNASE1 with regard to all the SNPs in wide-ranging ethnic groups could be first demonstrated. Further, there was no correlation of all the polymorphic SNPs other than nonsynonymous ones with serum DNase I activity levels. Polymorphic SNPs other than the exonic SNPs might not be directly related to common diseases through alterations in in vivo levels of the activity.     

IKKε regulates cell elongation through recycling endosome shuttling

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Highlights? Recycling endosomes shuttle along the proximal-distal axis of growing bristles ? IKK? locally antagonizes Rab11 effector Nuf at the bristle tip by phosphorylation ? Nuf-endosome pathway is independent of actin cytoskeleton or DIAP1-caspase pathway ? Mammalian IKK-related kinases antagonize Rab11-FIP3 through phosphorylation     

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.     

Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6-PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type-specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.     

Isolation and characterization of tetrachloroethylene- and <Emphasis Type="Italic">cis</Emphasis>-1,2-dichloroethylene-dechlorinating propionibacteria

Two rapidly growing propionibacteria that could reductively dechlorinate tetrachloroethylene (PCE) and cis-1,2-dichloroethylene (cis-DCE) to ethylene were isolated from environmental sediments. Metabolic characterization and partial sequence analysis of their 16S rRNA genes showed that the new isolates, designated as strains Propionibacterium sp. HK-1 and Propionibacterium sp. HK-3, did not match any known PCE- or cis-DCE-degrading bacteria. Both strains dechlorinated relatively high concentrations of PCE (0.3 mM) and cis-DCE (0.52 mM) under anaerobic conditions without accumulating toxic intermediates during incubation. Cell-free extracts of both strains catalyzed PCE and cis-DCE dechlorination; degradation was accelerated by the addition of various electron donors. PCE dehalogenase from strain HK-1 was mediated by a corrinoid protein, since the dehalogenase was inactivated by propyl iodide only after reduction by titanium citrate. The amounts of chloride ions (0.094 and 0.103 mM) released after PCE (0.026 mM) and cis-DCE (0.05 mM) dehalogenation using the cell-free enzyme extracts of both strains, HK-1 and HK-3, were stoichiometrically similar (91 and 100%), indicating that PCE and cis-DCE were fully dechlorinated. Radiotracer studies with [1,2-¹⁴C] PCE and [1,2-¹⁴C] cis-DCE indicated that ethylene was the terminal product; partial conversion to ethylene was observed. Various chlorinated aliphatic compounds (PCE, trichloroethylene, cis-DCE, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2-trichloroethane, and vinyl chloride) were degraded by cell-free extracts of strain HK-1.     

Alteration of plasma glutamate and glutamine levels in children with high-functioning autism

Background

It has recently been hypothesized that hyperglutamatergia in the brain is involved in the pathophysiology of autism. However, there is no conclusive evidence of the validity of this hypothesis. As peripheral glutamate/glutamine levels have been reported to be correlated with those of the central nervous system, the authors examined whether the levels of 25 amino acids, including glutamate and glutamine, in the platelet-poor plasma of drug-naïve, male children with high-functioning autism (HFA) would be altered compared with those of normal controls.

Methodology/Principal Findings

Plasma levels of 25 amino acids in male children (N = 23) with HFA and normally developed healthy male controls (N = 22) were determined using high-performance liquid chromatography. Multiple testing was allowed for in the analyses. Compared with the normal control group, the HFA group had higher levels of plasma glutamate and lower levels of plasma glutamine. No significant group difference was found in the remaining 23 amino acids. The effect size (Cohen''s d) for glutamate and glutamine was large: 1.13 and 1.36, respectively. Using discriminant analysis with logistic regression, the two values of plasma glutamate and glutamine were shown to well-differentiate the HFA group from the control group; the rate of correct classification was 91%.

Conclusions/Significance

The present study suggests that plasma glutamate and glutamine levels can serve as a diagnostic tool for the early detection of autism, especially normal IQ autism. These findings indicate that glutamatergic abnormalities in the brain may be associated with the pathobiology of autism.