Direct electrochemical regeneration of NADH from NAD+ using cholesterol-modified gold amalgam electrode

The efficiency of the direct electrochemical regeneration of NADH from NAD+ was enhanced by applying a cholesterol-modified gold amalgam electrode. The modified electrode was prepared by immersing gold plate in mercury and casting few drops of cholesteryl oleate solution over the gold amalgam. Coenzymatically active NADH was formed from NAD+ directly at the cholesterol-modified gold amalgam electrode which is supposed to hinder the dimerization of the NAD radicals on its membrane surface. The direct electrochemical NAD+ reduction process was used favorably to drive an enzymatic reduction of pyruvate to d-lactate. d-Lactate of 18.2 mm was obtained from pyruvate of 25.3 mm at 21 h of total reaction time in the electrolysis of 50 cm3 solution with the electrode of 6 cm2area. The turnover number for NAD+ was estimated as 1400.     

Induction of mouse Ca(2+)-sensitive chloride channel 2 gene during involution of mammary gland.

To understand molecular mechanisms that regulate mammary gland involution, we identified involution-induced cDNA clones by suppression subtractive hybridization methods. Nucleotide sequencing of a clone revealed that it was 97% identical to Ca(2+)-sensitive chloride channel 1 (mCLCA1) gene that has been identified in lung tissue. We concluded that our clone was derived from different gene with mCLCA1 and named it mCLCA2. We confirmed that expression of mCLCA2 gene was predominant in mammary gland while mCLCA1 mRNA was mainly detected in lung tissues by RT-PCR. Northern analysis showed that the mCLCA2 gene was induced at involution phase compared to pregnant and lactating phases of mammary gland. Under serum starvation, HC11 mammary epithelial cells showed DNA fragmentation and induction of mCLCA2 expression.     

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

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.