bluuebunny/crossref_metadata_2025 huggingface dataset:
RNAi Silencing of Genes for Elicitation or Biosynthesis of 5-Deoxyisoflavonoids Suppresses Race-Specific Resistance and Hypersensitive Cell Death in Phytophthora sojae Infected Tissues
Authors: Graham Terrence L. , Graham Madge Y. , Subramanian Senthil , Yu Oliver
Published: April 2007

Isoflavonoids are thought to play an important role in soybean (Glycine max) resistance to Phytophthora sojae. This was addressed by silencing two genes for their biosynthesis and a third gene controlling their elicitation. Silencing of genes for isoflavone synthase (IFS) or chalcone reductase (CHR) was achieved in soybean roots through an Agrobacterium rhizogenes-mediated RNAi approach. Effectiveness of silencing was followed both by quantitative reverse transcriptase-polymerase chain reaction and high-performance liquid chromatography analyses. Silencing either IFS or CHR led to a breakdown of Rps-mediated resistance to race 1 of P. sojae in ‘W79’ (Rps 1c) or ‘W82’ (Rps 1k) soybean. Loss of resistance was accompanied by suppression of hypersensitive (HR) cell death in both cultivars and suppression of cell death-associated activation of hydrogen peroxide and peroxidase. The various results suggest that the 5-deoxyisoflavonoids play a critical role in the establishment of cell death and race-specific resistance. The P. sojae cell wall glucan elicitor, a potent elicitor of 5-deoxyisoflavonoids, triggered a cell death response in roots that was also suppressed by silencing either CHR or IFS. Furthermore, silencing of the elicitor-releasing endoglucanase (PR-2) led to a loss of HR cell death and race-specific resistance to P. sojae and also to a loss of isoflavone and cell death responses to cell wall glucan elicitor. Taken together, these results suggest that in situ release of active fragments from a general resistance elicitor (pathogen-associated molecular pattern) is necessary for HR cell death in soybean roots carrying resistance genes at the Rps 1 locus, and that this cell death response is mediated through accumulations of the 5-deoxyisoflavones.
Rice NON-YELLOW COLORING1 Is Involved in Light-Harvesting Complex II and Grana Degradation during Leaf Senescence
Authors: Kusaba Makoto , Ito Hisashi , Morita Ryouhei , Iida Shuichi , Sato Yutaka , Fujimoto Masaru , Kawasaki Shinji , Tanaka Ryouichi , Hirochika Hirohiko , Nishimura Minoru , Tanaka Ayumi
Published: April 2007

Chlorophyll degradation is an aspect of leaf senescence, which is an active process to salvage nutrients from old tissues. non-yellow coloring1 (nyc1) is a rice (Oryza sativa) stay-green mutant in which chlorophyll degradation during senescence is impaired. Pigment analysis revealed that degradation of not only chlorophylls but also light-harvesting complex II (LHCII)–bound carotenoids was repressed in nyc1, in which most LHCII isoforms were selectively retained during senescence. Ultrastructural analysis of nyc1 chloroplasts revealed that large and thick grana were present even in the late stage of senescence, suggesting that degradation of LHCII is required for the proper degeneration of thylakoid membranes. Map-based cloning of NYC1 revealed that it encodes a chloroplast-localized short-chain dehydrogenase/reductase (SDR) with three transmembrane domains. The predicted structure of the NYC1 protein and the phenotype of the nyc1 mutant suggest the possibility that NYC1 is a chlorophyll b reductase. Although we were unable to detect the chlorophyll b reductase activity of NYC1, NOL (for NYC1-like), a protein closely related to NYC1 in rice, showed chlorophyll b reductase activity in vitro. We suggest that NYC1 and NOL encode chlorophyll b reductases with divergent functions. Our data collectively suggest that the identified SDR protein NYC1 plays essential roles in the regulation of LHCII and thylakoid membrane degradation during senescence.
Binding of Cyt1Aa and Cry11Aa Toxins of
Bacillus thuringiensis
Serovar
israelensis
to Brush Border Membrane Vesicles of
Tipula paludosa
(Diptera: Nematocera) and Subsequent Pore Formation
Authors: Oestergaard Jesko , Ehlers Ralf-Udo , Martínez-Ramírez Amparo C. , Real Maria Dolores
Published: April 2007

Bacillus thuringiensis
serovar
israelensis
(
B. thuringiensis
subsp.
israelensis
) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant
B. thuringiensis
subsp.
israelensis
strains expressing only one of the toxins was determined with first instars of
Tipula paludosa
(Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of
B. thuringiensis
subsp.
israelensis
ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of
T. paludosa
were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to
T. paludosa
BBMVs was detected, whereas the 32-kDa fragment bound to
T. paludosa
BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of
T. paludosa
, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in
T. paludosa
BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in
T. paludosa
.
Microbial Diversity in Natural Asphalts of the Rancho La Brea Tar Pits
Authors: Kim Jong-Shik , Crowley David E.
Published: April 2007

Bacteria commonly inhabit subsurface oil reservoirs, but almost nothing is known yet about microorganisms that live in naturally occurring terrestrial oil seeps and natural asphalts that are comprised of highly recalcitrant petroleum hydrocarbons. Here we report the first survey of microbial diversity in ca. 28,000-year-old samples of natural asphalts from the Rancho La Brea Tar Pits in Los Angeles, CA. Microbiological studies included analyses of 16S rRNA gene sequences and DNA encoding aromatic ring-hydroxylating dioxygenases from two tar pits differing in chemical composition. Our results revealed a wide range of phylogenetic groups within the
Archaea
and
Bacteria
domains, in which individual taxonomic clusters were comprised of sets of closely related species within novel genera and families. Fluorescent staining of asphalt-soil particles using phylogenetic probes for
Archaea
,
Bacteria
, and
Pseudomonas
showed coexistence of mixed microbial communities at high cell densities. Genes encoding dioxygenases included three novel clusters of enzymes. The discovery of life in the tar pits provides an avenue for further studies of the evolution of enzymes and catabolic pathways for bacteria that have been exposed to complex hydrocarbons for millennia. These bacteria also should have application for industrial microbiology and bioremediation.
Ammonia-Oxidizing β-Proteobacteria from the Oxygen Minimum Zone off Northern Chile
Authors: Molina Verónica , Ulloa Osvaldo , Farías Laura , Urrutia Homero , Ramírez Salvador , Junier Pilar , Witzel Karl-Paul
Published: April 2007

The composition of ammonia-oxidizing bacteria from the β-
Proteobacteria
subclass (βAOB) was studied in the surface and upper-oxycline oxic waters (2- to 50-m depth, ∼200 to 44 μM O
2
) and within the oxygen minimum zone (OMZ) suboxic waters (50- to 400-m depth, ≤10 μM O
2
) of the eastern South Pacific off northern Chile. This study was carried out through cloning and sequencing of genes coding for 16S rRNA and the ammonia monooxygenase enzyme active subunit (
amoA
). Sequences affiliated with
Nitrosospira
-like cluster 1 dominated the 16S rRNA gene clone libraries constructed from both oxic and suboxic waters. Cluster 1 consists exclusively of yet-uncultivated βAOB from marine environments. However, a single clone, out of 224 obtained from the OMZ, was found to belong to
Nitrosospira
lineage cluster 0. To our knowledge, cluster 0 sequences have been derived from βAOB isolated only from sand, soil, and freshwater environments. Sequences in clone libraries of the
amoA
gene from the surface and upper oxycline could be grouped in a marine subcluster, also containing no cultured representatives. In contrast, all 74
amoA
sequences originating from the OMZ were either closely affiliated with cultured
Nitrosospira
spp. from clusters 0 and 2 or with other yet-uncultured βAOB from soil and an aerated-anoxic Orbal process waste treatment plant. Our results reveal the presence of
Nitrosospira
-like βAOB in both oxic and suboxic waters associated with the OMZ but with a clear community shift at the functional level (
amoA
) along the strong oxygen gradient.
Phylogenetic Composition of Rocky Mountain Endolithic Microbial Ecosystems
Authors: Walker Jeffrey J. , Pace Norman R.
Published: April 2007

The endolithic environment, the pore space in rocks, is a ubiquitous microbial habitat. Photosynthesis-based endolithic communities inhabit the outer few millimeters to centimeters of rocks exposed to the surface. Such endolithic ecosystems have been proposed as simple, tractable models for understanding basic principles in microbial ecology. In order to test previously conceived hypotheses about endolithic ecosystems, we studied selected endolithic communities in the Rocky Mountain region of the United States with culture-independent molecular methods. Community compositions were determined by determining rRNA gene sequence contents, and communities were compared using statistical phylogenetic methods. The results indicate that endolithic ecosystems are seeded from a select, global metacommunity and form true ecological communities that are among the simplest microbial ecosystems known. Statistical analysis showed that biogeographical characteristics that control community composition, such as rock type, are more complex than predicted. Collectively, results of this study support the idea that patterns of microbial diversity found in endolithic communities are governed by principles similar to those observed in macroecological systems.
Detection and Genotyping of
Arcobacter
and
Campylobacter
Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays
Authors: Quiñones Beatriz , Parker Craig T. , Janda John M. , Miller William G. , Mandrell Robert E.
Published: April 2007

To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of
Arcobacter
and the presence of
Campylobacter
in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both
Arcobacter butzleri
and thermotolerant
Campylobacter jejuni
and
Campylobacter coli
. These microarrays showed a high level of probe specificity; the signal intensities detected for
A. butzleri
,
C. coli
, or
C. jejuni
probes were at least 10-fold higher than the background levels. Specific identification of
A. butzleri
,
C. coli
, and
C. jejuni
was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media,
C. jejuni
isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of
A. butzleri
and increased the recovery of
C. jejuni. C. jejuni
isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the
C. jejuni
isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000
C. jejuni
cells. Interestingly, the use of
C. jejuni
sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of
C. jejuni
in whole chicken carcass samples.
C. jejuni
was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.
Involvement of aMagnaporthe griseaSerine/Threonine Kinase Gene, MgATG1, in Appressorium Turgor and Pathogenesis
Authors: Liu Xiao-Hong , Lu Jian-Ping , Zhang Lei , Dong Bo , Min Hang , Lin Fu-Cheng
Published: April 2007

We isolated an MgATG1gene encoding a serine/threonine protein kinase from the rice blast fungusMagnaporthe grisea. In the ΔMgatg1mutant, in which the MgATG1gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the ΔMgatg1mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity inM. grisea.
Molecular Phylogenetics of
Candida albicans
Authors: Odds Frank C. , Bougnoux Marie-Elisabeth , Shaw Duncan J. , Bain Judith M. , Davidson Amanda D. , Diogo Dorothée , Jacobsen Mette D. , Lecomte Maud , Li Shu-Ying , Tavanti Arianna , Maiden Martin C. J. , Gow Neil A. R. , d'Enfert Christophe
Published: April 2007

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (
MAT
) status, and antifungal susceptibility for a panel of 1,391
Candida albicans
isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for
MAT
homozygous
a
/
a
types and then α/α types and was lowest for heterozygous
a
/α types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.
Further Characterization of the Signaling Proteolysis Step in theAspergillus nidulanspH Signal Transduction Pathway
Authors: Peñas María M. , Hervás-Aguilar América , Múnera-Huertas Tatiana , Reoyo Elena , Peñalva Miguel Á. , Arst Herbert N. , Tilburn Joan
Published: April 2007

TheAspergillus nidulanspH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved “signaling protease box” in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC3-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC3-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.
Genomic and Biochemical Analysis of Lipid Biosynthesis in the Unicellular Rhodophyte
Cyanidioschyzon merolae
: Lack of a Plastidic Desaturation Pathway Results in the Coupled Pathway of Galactolipid Synthesis
Authors: Sato Naoki , Moriyama Takashi
Published: April 2007

The acyl lipids making up the plastid membranes in plants and algae are highly enriched in polyunsaturated fatty acids and are synthesized by two distinct pathways, known as the prokaryotic and eukaryotic pathways, which are located within the plastids and the endoplasmic reticulum, respectively. Here we report the results of biochemical as well as genomic analyses of lipids and fatty acids in the unicellular rhodophyte
Cyanidioschyzon merolae
. All of the glycerolipids usually found in photosynthetic algae were found, such as mono- and digalactosyl diacylglycerol, sulfolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. However, the fatty acid composition was extremely simple. Only palmitic, stearic, oleic, and linoleic acids were found as major acids. In addition, 3-
trans
-hexadecanoic acid was found as a very minor component in phosphatidylglycerol. Unlike the case for most other photosynthetic eukaryotes, polyenoic fatty acids having three or more double bonds were not detected. These results suggest that polyunsaturated fatty acids are not necessary for photosynthesis in eukaryotes. Genomic analysis suggested that
C. merolae
lacks acyl lipid desaturases of cyanobacterial origin as well as stearoyl acyl carrier protein desaturase, both of which are major desaturases in plants and green algae. The results of labeling experiments with radioactive acetate showed that the desaturation leading to linoleic acid synthesis occurs on phosphatidylcholine located outside the plastids. Monogalactosyl diacylglycerol is therefore synthesized by the coupled pathway, using plastid-derived palmitic acid and endoplasmic reticulum-derived linoleic acid. These results highlight essential differences in lipid biosynthetic pathways between the red algae and the green lineage, which includes plants and green algae.
Genome of
Methylobacillus flagellatus
, Molecular Basis for Obligate Methylotrophy, and Polyphyletic Origin of Methylotrophy
Authors: Chistoserdova Ludmila , Lapidus Alla , Han Cliff , Goodwin Lynne , Saunders Liz , Brettin Tom , Tapia Roxanne , Gilna Paul , Lucas Susan , Richardson Paul M. , Lidstrom Mary E.
Published: April 2007

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer,
Methylobacillus flagellatus
(strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of
M. flagellatus
was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs,
Methylobacterium extorquens
(an alphaproteobacterium, 7 Mbp),
Methylibium petroleiphilum
(a betaproteobacterium, 4 Mbp), and
Methylococcus capsulatus
(a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in
M. flagellatus
were more similar to those in
M. capsulatus
and
M. extorquens
than to the ones in the more closely related
M. petroleiphilum
species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in
Betaproteobacteria
.
Colicin E2 Is Still in Contact with Its Receptor and Import Machinery When Its Nuclease Domain Enters the Cytoplasm
Authors: Duché Denis
Published: April 2007

Colicins reach their targets in susceptible
Escherichia coli
strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Colicin E2 (ColE2) is a cytotoxic protein that recognizes the outer membrane receptor BtuB. After gaining access to the cytoplasmic membrane of sensitive
Escherichia coli
cells, ColE2 enters the cytoplasm to cleave DNA. After binding to BtuB, ColE2 interacts with the Tol system to reach its target. However, it is not known if the entire colicin or only the nuclease domain of ColE2 enters the cell. Here I show that preincubation of ColE2 with
Escherichia coli
cells prevents binding and translocation of pore-forming colicins of group A but not of group B. This inhibition persisted even when cells were incubated with ColE2 for 30 min before the addition of pore-forming colicins, indicating that ColE2 releases neither its receptor nor its translocation machinery when its nuclease domain enters the cells. These competition experiments enabled me to estimate the time required for ColE2 binding to its receptor and translocation.
SOS Induction in a Subpopulation of Structural Maintenance of Chromosome (Smc) Mutant Cells in
Bacillus subtilis
Authors: Britton Robert A. , Küster-Schöck Elke , Auchtung Thomas A. , Grossman Alan D.
Published: April 2007

The structural maintenance of chromosome (Smc) protein is highly conserved and involved in chromosome compaction, cohesion, and other DNA-related processes. In
Bacillus subtilis
,
smc
null mutations cause defects in DNA supercoiling, chromosome compaction, and chromosome partitioning. We investigated the effects of
smc
mutations on global gene expression in
B. subtilis
using DNA microarrays. We found that an
smc
null mutation caused partial induction of the SOS response, including induction of the defective prophage PBSX. Analysis of SOS and phage gene expression in single cells indicated that approximately 1% of
smc
mutants have fully induced SOS and PBSX gene expression while the other 99% of cells appear to have little or no expression. We found that induction of PBSX was not responsible for the chromosome partitioning or compaction defects of
smc
mutants. Similar inductions of the SOS response and PBSX were observed in cells depleted of topoisomerase I, an enzyme that relaxes negatively supercoiled DNA.
Citrate Sensing by the C
4
-Dicarboxylate/Citrate Sensor Kinase DcuS of
Escherichia coli
: Binding Site and Conversion of DcuS to a C
4
-Dicarboxylate- or Citrate-Specific Sensor
Authors: Krämer J. , Fischer J. D. , Zientz E. , Vijayan V. , Griesinger C. , Lupas A. , Unden G.
Published: April 2007

The histidine protein kinase DcuS of
Escherichia coli
senses C
4
-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C
4
-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C
4
-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C
4
-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C
4
-dicarboxylate-specific sensor (DcuS
DC
) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS
Cit
). DcuS
DC
and DcuS
Cit
had complementary effector specificities and responded either to C
4
-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C
4
-dicarboxylates) via the C
4
-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C
4
-dicarboxylates; sites C1 and H are required for other essential purposes.
Role of RNA Structure and Susceptibility to RNase E in Regulation of a Cold Shock mRNA,
cspA
mRNA
Authors: Hankins Janet S. , Zappavigna Christopher , Prud'homme-Généreux Annie , Mackie George A.
Published: April 2007

Degradation of the
cspA
mRNA in vivo is very rapid at temperatures greater than 30°C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the
cspA
mRNA preferentially at a single site in vitro between two stem-loops ∼24 residues 3′ to the termination codon and ∼31 residues from the 3′ end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5′ end of
cspA
mRNA. A 5′ stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the
cspA
mRNA.
Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Valladares Ana , Maldener Iris , Muro-Pastor Alicia M. , Flores Enrique , Herrero Antonia
Published: April 2007

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium
Anabaena
sp. strain PCC 7120 bearing inactivated
cox2
and/or
cox3
genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the
cox2 cox3
double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes
nifHDK
and
fdxH
. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (
cox3
) and undetectable in strain CSAV141 (
cox2 cox3
). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N
2
fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of
nif
genes in
Anabaena
seems not to be affected by oxygen.
Molecular Analysis of Genes in
Nostoc punctiforme
Involved in Pilus Biogenesis and Plant Infection
Authors: Duggan Paula S. , Gottardello Priscila , Adams David G.
Published: April 2007

Hormogonia are the infective agents in many cyanobacterium-plant symbioses. Pilus-like appendages are expressed on the hormogonium surface, and mutations in
pil
-like genes altered surface piliation and reduced symbiotic competency. This is the first molecular evidence that pilus biogenesis in a filamentous cyanobacterium requires a type IV pilus system.
Predation by
Bdellovibrio bacteriovorus
HD100 Requires Type IV Pili
Authors: Evans Katy J. , Lambert Carey , Sockett R. Elizabeth
Published: April 2007

Early electron microscopy and more recent studies in our laboratory of
Bdellovibrio bacteriovorus
cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the
B. bacteriovorus
HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of
B. bacteriovorus
HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The
pilA
gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple
Bdellovibrio
replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of
pilA
in replicate isolates abolished
Bdellovibrio
predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of
pil
genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory
Bdellovibrio
throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in
Bdellovibrio
predation.
Molecular basis for PP2A regulatory subunit B56α targeting in cardiomyocytes
Authors: Bhasin Naina , Cunha Shane R. , Mudannayake Malkanthi , Gigena Marisa S. , Rogers Terry B. , Mohler Peter J.
Published: April 2007

Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of β-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56α is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56α are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56α for ankyrin-B association as a 13 residue motif in the B56α COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B+/−cardiomyocytes results in disorganized distribution of B56α that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides.
Tumor necrosis factor-α is toxic via receptor 1 and protective via receptor 2 in a murine model of myocardial infarction
Authors: Monden Yoshiya , Kubota Toru , Inoue Takahiro , Tsutsumi Takaki , Kawano Shunichi , Ide Tomomi , Tsutsui Hiroyuki , Sunagawa Kenji
Published: April 2007

Tumor necrosis factor (TNF)-α induced in damaged myocardium has been considered to be cardiotoxic. TNF-α initiates its biological effects by binding two distinct receptors: R1 (p55) and R2 (p75). Although TNF-α has been shown to be cardiotoxic via R1-mediated pathways, little is known about the roles of R2-mediated pathways in myocardial infarction (MI). We created MI in R1 knockout (R1KO), R2KO, and wild-type (WT) mice by ligating the left coronary artery. Functional, histological, and biochemical analyses were performed 4 wk after ligation. Although infarct size was not different among WT, R1KO, and R2KO mice, post-MI survival was significantly improved in R1KO but not R2KO mice. R1KO significantly ameliorated contractile dysfunction after MI, whereas R2KO significantly exaggerated ventricular dilatation and dysfunction. Myocyte hypertrophy and interstitial fibrosis in noninfarct myocardium was exacerbated in R2KO but not in R1KO mice. Expression of R1, which was not affected by MI and was nullified in R1KO mice, was significantly upregulated in R2KO mice. In contrast, expression of R2, which was significantly upregulated by MI and was nullified in R2KO mice, was unaffected in R1KO mice. Meanwhile, TNF-α expression, which was significantly upregulated in noninfarct myocardium after MI, was not affected by R1KO or R2KO. However, transcript levels of IL-6, IL-1β, transforming growth factor-β, and monocyte chemotactic protein-1, which were significantly upregulated after MI, were significantly downregulated in R1KO mice. In contrast, transcript levels of IL-6 and IL-1β were significantly further upregulated in R2KO mice. TNF-α is toxic via R1 and protective via R2 in a murine model of MI. Selective blockade of R1 may be a candidate therapeutic intervention for MI.
TNF-α blockade decreases oxidative stress in the paraventricular nucleus and attenuates sympathoexcitation in heart failure rats
Authors: Guggilam Anuradha , Haque Masudul , Kerut Edmund Kenneth , McIlwain Elizabeth , Lucchesi Pamela , Seghal Inder , Francis Joseph
Published: April 2007

Oxidative stress plays an important role in the pathophysiology of cardiovascular disease. Recent evidence suggests that cytokines induce oxidative stress and contribute to cardiac dysfunction. In this study, we investigated whether increased circulating and tissue levels of tumor necrosis factor (TNF)-α in congestive heart failure (CHF) modulate the expression of NAD(P)H oxidase subunits, Nox2 and its isoforms, in the paraventricular nucleus (PVN) of the hypothalamus and contribute to exaggerated sympathetic drive in CHF. Heart failure was induced in Sprague-Dawly rats by coronary artery ligation and was confirmed using echocardiography. Pentoxifylline (PTX) was used to block the production of cytokines for a period of 5 wk. CHF induced a significant increase in the production of reactive oxygen species (ROS) in the left ventricle (LV) and in the PVN. The mRNA and protein expression of TNF-α, Nox1, Nox2, and Nox4 was significantly increased in the LV and PVN of CHF rats. CHF also decreased ejection fraction, increased Tei index, and increased circulating catecholamines (epinephrine and norepinephrine) and renal sympathetic activity (RSNA). In contrast, treatment with PTX in CHF rats completely blocked oxidative stress and decreased the production of TNF-α and Nox2 isoforms both in the LV and PVN. PTX treatment also decreased catecholamines and RSNA and prevented further decrease in cardiac function. In summary, TNF-α blockade attenuates ROS and sympathoexcitation in CHF. This study unveils new mechanisms by which cytokines play a role in the pathogenesis of CHF, thus underscoring the importance of targeting cytokines in heart failure.
Enhanced activity of ventricular Na+-HCO3− cotransport in pressure overload hypertrophy
Authors: Yamamoto Taku , Shirayama Takeshi , Sakatani Tomohiko , Takahashi Tomosaburo , Tanaka Hideo , Takamatsu Tetsuro , Spitzer Kenneth W. , Matsubara Hiroaki
Published: April 2007

The Na+-HCO3− cotransporter (NBC) plays a key role in intracellular pH (pHi) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pHi was measured in single cells using the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)5-( 6 )carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ( JNBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na+/H+ exchange was also increased in hypertrophied myocytes, the relative enhancement of JNBC was larger, 6) membrane depolarization markedly increased JNBC in hypertrophied myocytes, and 7) losartan, an ANG II AT1 receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na+ overload, which may render the ventricles more vulnerable to Ca2+ overload during ischemia-reperfusion.
Cardiomyocyte-restricted restoration of nitric oxide synthase 3 attenuates left ventricular remodeling after chronic pressure overload
Authors: Buys Emmanuel S. , Raher Michael J. , Blake Sarah L. , Neilan Tomas G. , Graveline Amanda R. , Passeri Jonathan J. , Llano Miguel , Perez-Sanz Teresa M. , Ichinose Fumito , Janssens Stefan , Zapol Warren M. , Picard Michael H. , Bloch Kenneth D. , Scherrer-Crosbie Marielle
Published: April 2007

Although nitric oxide synthase (NOS)3 is implicated as an important modulator of left ventricular (LV) remodeling, its role in the cardiac response to chronic pressure overload is controversial. We examined whether selective restoration of NOS3 to the hearts of NOS3-deficient mice would modulate the LV remodeling response to transverse aortic constriction (TAC). LV structure and function were compared at baseline and after TAC in NOS3-deficient (NOS3−/−) mice and NOS3−/− mice carrying a transgene directing NOS3 expression specifically in cardiomyocytes (NOS3−/−TG mice). At baseline, echocardiographic assessment of LV dimensions and function, invasive hemodynamic measurements, LV mass, and myocyte width did not differ between the two genotypes. Four weeks after TAC, echocardiographic and hemodynamic indexes of LV systolic function indicated that contractile performance was better preserved in NOS3−/−TG mice than in NOS3−/− mice. Echocardiographic LV wall thickness and cardiomyocyte width were greater in NOS3−/− mice than in NOS3−/−TG mice. TAC-induced cardiac fibrosis did not differ between these genotypes. TAC increased cardiac superoxide generation in NOS3−/−TG but not NOS3−/− mice. The ratio of NOS3 dimers to monomers did not differ before and after TAC in NOS3−/−TG mice. Restoration of NOS3 to the heart of NOS3-deficient mice attenuates LV hypertrophy and dysfunction after TAC, suggesting that NOS3 protects against the adverse LV remodeling induced by prolonged pressure overload.
Transplanted embryonic stem cells following mouse myocardial infarction inhibit apoptosis and cardiac remodeling
Authors: Singla Dinender K. , Lyons Gary E. , Kamp Timothy J.
Published: April 2007

We have previously shown that mouse embryonic stem (ES) cells transplanted following myocardial infarction (MI) differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration was relatively meager compared with the observed functional improvement. Therefore, we hypothesize that mechanisms in addition to regeneration contribute to the functional improvement from ES cell therapy. In this study, we examined the effect of mouse ES cells transplanted post-MI on cardiac apoptosis, fibrosis, and hypertrophy. MI was produced by left coronary artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing enhanced green fluorescent protein and β-galactosidase, respectively, were tested. Post-MI intramyocardial injection of 3 × 104 ES cells was compared with injection of medium alone. Terminal deoxynucleotidyl nick end labeling (TUNEL), immunofluorescence, and histology were used to examine the effect of transplanted ES cells on apoptosis, fibrosis, and hypertrophy. Two weeks post-MI, ES cell-transplanted hearts exhibited a significant decrease in TUNEL-stained nuclei (mean ± SE; MI+medium = 12 ± 1.5%; MI+ES cells = 6.6 ± 1%, P < 0.05). TUNEL-positive nuclei were confirmed to be apoptotic by colabeling with a caspase-3 antibody. Cardiac fibrosis was 57% less in the MI+ES cell group compared with the MI + medium group ( P < 0.05) as shown with Masson's trichrome staining. Picrosirius red staining confirmed a decreased amount of collagen present in the MI+ES cell group. Cardiomyocyte hypertrophy was significantly decreased following ES cell transplantation compared with medium control animals. In conclusion, transplanted mouse ES cells in the infarcted heart inhibit apoptosis, fibrosis, and hypertrophy, thereby reducing adverse remodeling.
Increased mitochondrial H2O2 production promotes endothelial NF-κB activation in aged rat arteries
Authors: Ungvari Zoltan , Orosz Zsuzsanna , Labinskyy Nazar , Rivera Aracelie , Xiangmin Zhao , Smith Kira , Csiszar Anna
Published: April 2007

Previous studies have shown that the aging vascular system undergoes pro-atherogenic phenotypic changes, including increased oxidative stress and a pro-inflammatory shift in endothelial gene expression profile. To elucidate the link between increased oxidative stress and vascular inflammation in aging, we compared the carotid arteries and aortas of young and aged (24 mo old) Fisher 344 rats. In aged vessels there was an increased NF-κB activity (assessed by luciferase reporter gene assay and NF-κB binding assay), which was attenuated by scavenging H2O2. Aging did not alter the vascular mRNA and protein expression of p65 and p50 subunits of NF-κB. In endothelial cells of aged vessels there was an increased production of H2O2 (assessed by 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-acetyl ester fluorescence), which was attenuated by the mitochondrial uncoupler FCCP. In young arteries and cultured endothelial cells, antimycin A plus succinate significantly increased FCCP-sensitive mitochondrial H2O2 generation, which was associated with activation of NF-κB. In aged vessels inhibition of NF-κB (by pyrrolidenedithiocarbamate, resveratrol) significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness. Thus increased mitochondrial oxidative stress contributes to endothelial NF-κB activation, which contributes to the pro-inflammatory phenotypic alterations in the aged vaculature. Our model predicts that by reducing mitochondrial H2O2 production and/or directly inhibiting NF-κB novel anti-aging pharmacological treatments (e.g., calorie restriction mimetics) will exert significant anti-inflammatory and vasoprotective effects.
Human recombinant chromogranin A-derived vasostatin-1 mimics preconditioning via an adenosine/nitric oxide signaling mechanism
Authors: Cappello Sandra , Angelone Tommaso , Tota Bruno , Pagliaro Pasquale , Penna Claudia , Rastaldo Raffaella , Corti Angelo , Losano Gianni , Cerra Maria Carmela
Published: April 2007

The acidic protein chromogranin A (CgA) is the precursor of several regulatory peptides generated by specific proteolytic processes. Human recombinant CgA NH2-terminal fragment STA-CgA1-78 (hrSTA-CgA1-78), containing vasostatin-1 (CgA1-76) domain, exerts a negative inotropic effect and counteracts the β-adrenergic positive inotropic effect on the rat heart. We hypothesized an involvement of nitric oxide (NO)-dependent pathway in both cardiodepression and cardioprotection by hrSTA-CgA1-78. We also hypothesized an involvement of adenosine A1 receptor and protein kinase C (PKC) in cardioprotection by hrSTA-CgA1-78. Therefore, we evaluated whether 1) the cardioinhibition mediated by hrSTA-CgA1-78 involves the Gi/o proteins/NO-dependent signal transduction cascade, 2) hrSTA-CgA1-78 induces ischemic preconditioning-like protective effects on the myocardium, and 3) inhibition of NO synthase (NOS), adenosine A1 receptor, or PKC affects hrSTA-CgA1-78 protection. Using the isolated rat heart, we found that the reduction of left ventricular pressure (LVP), rate-pressure product, and maximal values of the first derivative of LVP elicited by hrSTA-CgA1-78 at 33 nM is abolished by blocking Gi/o proteins with pertussis toxin, scavenging NO with hemoglobin, and blocking NOS activity with NG-monomethyl-l-arginine or N5-(iminoethyl)-l-ornithine, soluble guanylate cyclase with 1 H-[1,2,4]oxadiazole-[4,4-a]quinoxalin-1-one, and protein kinase (PKG) with KT5823. Data suggest the involvement of the Gi/o proteins/NO-cGMP-PKG pathway in the hrSTA-CgA1-78-dependent cardioinhibition. When given before 30 min of ischemia, hrSTA-CgA1-78 significantly reduced the size of the infarct from 64 ± 4 to 32 ± 3% of the left ventricular mass. This protective effect was abolished by either NOS inhibition or PKC blockade and was attenuated, but not suppressed, by the blockade of A1 receptors. These results suggest that hrSTA-CgA1-78 activity triggers two different pathways: one of these pathways is mediated by A1 receptors, and the other is mediated by NO release. As with repeated brief preconditioning ischemia, hrSTA-CgA1-78 may be considered a stimulus strong enough to trigger both pathways, which may converge on PKC.