Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca²⁺-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca²⁺ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Ca²⁺-dependent protein kinase 29 directly phosphorylates the hydrophilic loop of PIN-FORMED proteins to modulate their intracellular trafficking and Arabidopsis development.     

Introduction of tyramide signal amplification (TSA) to pre-embedding nanogold-silver staining at the electron microscopic level.

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.     

Diversity of bacterial community in freshwater of Woopo wetland

Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Genetic Subdivision of Chemosynthetic Endosymbionts of Solemya velum along the Southern New England Coast

Population-level genetic diversity in the obligate symbiosis between the bivalve Solemya velum and its thioautotrophic bacterial endosymbiont was examined. Distinct populations along the New England coast shared a single mitochondrial genotype but were fixed for unique symbiont genotypes, indicating high levels of symbiont genetic structuring and potential symbiont-host decoupling.Studies of endosymbioses between marine invertebrates and sulfur-oxidizing chemosynthetic bacteria have yielded tremendous insight into the biology of bacterium-eukaryote interactions. Though best described for deep-sea vents and cold seeps, these mutualisms, in which symbiont thioautotrophy supports the nutrition of both partners, are also ubiquitous in coastal sediments (17). Our understanding of these interactions stems largely from studies of symbioses involving protobranch bivalves in the family Solemyidae (16). Though solemyids and other species that form chemosynthetic symbioses occur globally, little is known about how symbionts and hosts are structured genetically across distinct populations. Characterizing these patterns is critical for understanding how symbiosis drives the coevolution of interacting species, as well as how environmental heterogeneity and dispersal affect local adaptation. This study examines the geographic structure of genetic variation in the symbiosis between chemosynthetic bacteria and the Atlantic protobranch Solemya velum.Solemya velum is ideal for studying the evolution of highly coadapted bacterium-eukaryote mutualisms. This small bivalve (∼1.5 to 3 cm) burrows in sulfide-rich coastal sediments, where it obtains most of its nutrition from thioautotrophic bacteria living within specialized gill cells (1, 10). Though observed from Florida to Canada (20), the distribution of S. velum is highly patchy, with seemingly suitable habitat often devoid of individuals (12). Consequently, molecular characterizations of this symbiosis have focused primarily on stable and locally abundant populations near Woods Hole, MA. Direct sequencing of the symbiont 16S rRNA gene from these individuals has revealed a single, unique phylotype clustering within the Gammaproteobacteria (5, 6, 9). DNA from this symbiont has been extracted from S. velum ovarian tissue, raising the hypothesis that symbionts are transmitted vertically from mother to offspring (11) and are therefore tightly coupled to the host''s life cycle and evolutionary history.If symbiont acquisition is strictly vertical in Solemya populations, the genealogies of the symbiont and the cotransmitted host mitochondrion should diverge in parallel (cospeciation) (8, 15, 18). However, lateral acquisition involving either symbiont uptake from the environment or horizontal transfer between co-occurring hosts has not been ruled out for Solemya populations and could decouple symbiont and host genealogies (18). Indeed, 16S phylogenies show that symbionts of diverse Solemya species are polyphyletic, a pattern inconsistent with the putative monophyly of the hosts (based on nonmolecular characters) and suggestive of multiple evolutionary origins (2, 9, 16). However, tests for symbiont-host codiversification below the species level in S. velum are lacking; sequence data from multiple populations will help resolve questions of cospeciation and symbiont transmission in this group.Here, distinct Solemya velum populations were genotyped to examine how symbiont diversity covaries with host diversity and geography. Individual bivalves (n = 12 to 22 per site) were collected from mudflats at four sites along the southern New England coast (Fig. ​(Fig.1A).1A). DNA was extracted from the symbiont-containing gills and used for PCR amplification of fragments of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the symbiont 16S gene and hypervariable internal transcribed spacer (16S-ITS) (Table ​(Table1;1; also see the supplemental material). Unambiguous contigs of 340 nucleotides (nt) for the COI locus and 716 nt for the 16S-ITS locus, including 241 nt of the 16S and 475 nt (∼95%) of the ITS, were generated via bidirectional direct sequencing of amplicons using BigDye chemistry. Symbiont identity was confirmed by blasting the 16S-ITS (Woods Hole [WH] phylotype) against an assembly of the S. velum symbiont genome from the same population (C. Cavanaugh, unpublished data). Blastn returned a single full-length hit with 100% identity across the locus. Genotype networks were then inferred via statistical parsimony in the program TCS (3).Open in a separate windowFIG. 1.(A) Locations of Solemya velum collection sites (stars) along the Atlantic Coast were Naushon Island, Woods Hole, MA (WH; 41.514°N, −70.712°W); Lake Tashmoo, Martha''s Vineyard, MA (MV; 41.465°N, −70.623°W); Judith Pond, RI (RI; 41.380°N, −71.502°W); and Shark River Island, NJ (NJ; 40.186°N, −74.030°W). (B) Parsimony networks of host COI and symbiont 16S-ITS genotypes. Open circle, single-nucleotide substitution in either the host COI (top; 340 nt) or symbiont 16S (241 nt); filled circle, single-nucleotide substitution in the ITS portion (475 nt) of the 16S-ITS sequence fragment (716 nt total); diagonal bar, single-nucleotide indel in the symbiont ITS; gen1 and gen2, genotypes 1 and 2. Values in parentheses show the number of S. velum individuals from which sequences were obtained at each site.

TABLE 1.

Symbiont and host primers used in PCR^a and direct sequencing

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.