bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Cloning and Characterization of Two NADH-Dependent 3-Quinuclidinone Reductases from Microbacterium luteolum JCM 9174
Authors: Isotani Kentaro , Kurokawa Junji , Suzuki Fumiko , Nomoto Syunsuke , Negishi Takashi , Matsuda Michiko , Itoh Nobuya 
Published: December 2012

ABSTRACT

We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (
R
)-(−)-3-quinuclidinol.
Microbacterium luteolum
JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from
M. luteolum
JCM 9174. The
bacC
gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The
qnr
gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in
Escherichia coli
as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (
R
)-(−)-3-quinuclidinol.
Comparison of Filters for Concentrating Microbial Indicators and Pathogens in Lake Water Samples
Authors: Francy Donna S. , Stelzer Erin A. , Brady Amie M. G. , Huitger Carrie , Bushon Rebecca N. , Ip Hon S. , Ware Michael W. , Villegas Eric N. , Gallardo Vicente , Lindquist H. D. Alan 
Published: December 2012

ABSTRACT

Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for
Escherichia coli
(68.3%) and
Cryptosporidium
(54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for
Enterococcus faecalis
(80.5%), avian influenza virus (0.02%), and
Giardia
(57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.
Soil Microbe Active Community Composition and Capability of Responding to Litter Addition after 12 Years of No Inputs
Authors: Yarwood Stephanie , Brewer Elizabeth , Yarwood Rockie , Lajtha Kate , Myrold David 
Published: December 2012

ABSTRACTOne explanation given for the high microbial diversity found in soils is that they contain a large inactive biomass that is able to persist in soils for long periods of time. This persistent microbial fraction may help to buffer the functionality of the soil community during times of low nutrients by providing a reservoir of specialized functions that can be reactivated when conditions improve. A study was designed to test the hypothesis: in soils lacking fresh root or detrital inputs, microbial community composition may persist relatively unchanged. Upon addition of new inputs, this community will be stimulated to grow and break down litter similarly to control soils. Soils from two of the Detrital Input and Removal Treatments (DIRT) at the H. J. Andrews Experimental Forest, the no-input and control treatment plots, were used in a microcosm experiment where Douglas-fir needles were added to soils. After 3 and 151 days of incubation, soil microbial DNA and RNA was extracted and characterized using quantitative PCR (qPCR) and 454 pyrosequencing. The abundance of 16S and 28S gene copies and RNA copies did not vary with soil type or amendment; however, treatment differences were observed in the abundance of archaeal ammonia-oxidizingamoAgene abundance. Analysis of ∼110,000 bacterial sequences showed a significant change in the active (RNA-based) community between day 3 and day 151, but microbial composition was similar between soil types. These results show that even after 12 years of plant litter exclusion, the legacy of community composition was well buffered against a dramatic disturbance.
Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae
Authors: Ha Suk-Jin , Kim Heejin , Lin Yuping , Jang Myoung-Uoon , Galazka Jonathan M. , Kim Tae-Jip , Cate Jamie H. D. , Jin Yong-Su 
Published: December 2012

ABSTRACT

Saccharomyces cerevisiae
cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (
cdt-1
) and intracellular β-glucosidase (
gh1-1
) genes from
Neurospora crassa
. Here, we report that an engineered
S. cerevisiae
strain expressing the putative hexose transporter gene
HXT2.4
from
Scheffersomyces stipitis
and
gh1-1
can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when
HXT2.4
is overexpressed in
S. cerevisiae
. However, cellobiose fermentation by the engineered strain expressing
HXT2.4
and
gh1-1
was much slower and less efficient than that by an engineered strain that initially expressed
cdt-1
and
gh1-1
. The rate of cellobiose fermentation by the
HXT2.4
-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved
S. cerevisiae
strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher

K
m

and 4-fold higher
V
max
values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in
S. cerevisiae
are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered
S. cerevisiae
strains.
Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii
Authors: Su Xiaoyun , Han Yejun , Dodd Dylan , Moon Young Hwan , Yoshida Shosuke , Mackie Roderick I. , Cann Isaac K. O. 
Published: December 2012

ABSTRACT

Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium
Caldicellulosiruptor bescii
were expressed in
Escherichia coli
, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated
k
cat
values of ∼8,000 and ∼4,500 s
−1
, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The
C. bescii
xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.
Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool
Authors: Loza Virginia , Perona Elvira , Mateo Pilar 
Published: December 2012

ABSTRACTBenthic cyanobacterial communities from Guadarrama River (Spain) biofilms were examined using temperature gradient gel electrophoresis (TGGE), comparing the results with microscopic analyses of field-fixed samples and the genetic characterization of cultured isolates from the river. Changes in the structure and composition of cyanobacterial communities and their possible association with eutrophication in the river downstream were studied by examining complex TGGE patterns, band extraction, and subsequent sequencing of 16S rRNA gene fragments. Band profiles differed among sampling sites depending on differences in water quality. The results showed that TGGE band richness decreased in a downstream direction, and there was a clear clustering of phylotypes on the basis of their origins from different locations according to their ecological requirements. Multivariate analyses (cluster analysis and canonical correspondence analysis) corroborated these differences. Results were consistent with those obtained from microscopic observations of field-fixed samples. According to the phylogenetic analysis, morphotypes observed in natural samples were the most common phylotypes in the TGGE sequences. These phylotypes were closely related toChamaesiphon,Aphanocapsa,Pleurocapsa,Cyanobium,Pseudanabaena,Phormidium, andLeptolyngbya. Differences in the populations in response to environmental variables, principally nutrient concentrations (dissolved inorganic nitrogen and soluble reactive phosphorus), were found. Some phylotypes were associated with low nutrient concentrations and high levels of dissolved oxygen, while other phylotypes were associated with eutrophic-hypertrophic conditions. These results support the view that once a community has been characterized and its genetic fingerprint obtained, this technique could be used for the purpose of monitoring rivers.
Protective Effects of Lactobacillus plantarum CCFM8610 against Acute Cadmium Toxicity in Mice
Authors: Zhai Qixiao , Wang Gang , Zhao Jianxin , Liu Xiaoming , Tian Fengwei , Zhang Hao , Chen Wei 
Published: December 2012

ABSTRACT

This study evaluated the protective effects of
Lactobacillus plantarum
CCFM8610, a selected probiotic with good cadmium binding capacity, against acute cadmium toxicity in mice. Ninety mice were divided into prevention and therapy groups. In the prevention groups, CCFM8610 was administered at 10
9
CFU once daily for 7 days, followed by a single oral dose of cadmium chloride at 1.8 mg cadmium for each mouse. In the therapy groups, the same dose of CCFM8610 was administered for 2 days after an identical single dose of cadmium exposure. Mice that received neither cadmium nor culture or that received cadmium alone served as negative and positive controls, respectively. The effects of both living and dead CCFM8610 on cadmium ion concentrations in feces, liver, and kidney were determined. Moreover, the alterations in reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and histopathology in the liver and kidney were investigated. The results showed that compared to the mice that received cadmium only, CCFM8610 treatment can effectively decrease intestinal cadmium absorption, reduce tissue cadmium accumulation, alleviate renal and hepatic oxidative stress, and ameliorate hepatic histopathological changes. Living CCFM8610 administered after cadmium exposure offered the most significant protection. Our results suggested that CCFM8610 is more effective against acute cadmium toxicity than a simple antioxidant treatment due to its special physiological functions and that it can be considered a new dietary therapeutic strategy against acute cadmium toxicity.
Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus
Authors: Samantaray Sweta , Neubauer Michael , Helmschrott Christoph , Wagener Johannes 
Published: December 2012

ABSTRACT

Aspergillus fumigatus
is a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity of
A. fumigatus
. A conditional
rom2
mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the Δ
wsc1
mutant and the Congo red, calcofluor white, and heat sensitivity of the Δ
midA
mutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.
MAPK-Activated Protein Kinase 2 Contributes to Clostridium difficile-Associated Inflammation
Authors: Bobo Linda D. , El Feghaly Rana E. , Chen Yee-Shiuan , Dubberke Erik R. , Han Zhuolin , Baker Alexandra H. , Li Jinmei , Burnham Carey-Ann D. , Haslam David B. 
Published: December 2012

ABSTRACT

Clostridium difficile
infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that
C. difficile
toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the
C. difficile
toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxin-induced IL-8 and GROα release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of
C. difficile-
infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic
C. difficile
among 100 patient stool samples submitted for
C. difficile
testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI.
Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase
Authors: Maeda Kazuhiko , Nagata Hideki , Kuboniwa Masae , Ojima Miki , Osaki Tsukasa , Minamino Naoto , Amano Atsuo 
Published: December 2012

ABSTRACT

Coaggregation of
Porphyromonas gingivalis
and oral streptococci is thought to play an important role in
P. gingivalis
colonization. Previously, we reported that
P. gingivalis
major fimbriae interacted with
Streptococcus oralis
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward
P. gingivalis
fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun.
77:
5130–5138, 2009). The present study aimed to identify and characterize
P. gingivalis
components other than fimbriae that interact with
S. oralis
GAPDH. A pulldown assay was performed to detect potential interactions between
P. gingivalis
client proteins and
S. oralis
recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely,
tonB
-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and
S. oralis
GAPDH were analyzed with a biomolecular interaction analysis system.
S. oralis
GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between
P. gingivalis
and
S. oralis
were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of
luxS
in
P. gingivalis
was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five
P. gingivalis
client proteins function as regulators in
P. gingivalis
biofilm formation with oral streptococci.
Enhanced Susceptibility to Citrobacter rodentium Infection in MicroRNA-155-Deficient Mice
Authors: Clare Simon , John Victoria , Walker Alan W. , Hill Jennifer L. , Abreu-Goodger Cei , Hale Christine , Goulding David , Lawley Trevor D. , Mastroeni Pietro , Frankel Gadi , Enright Anton J. , Vigorito Elena , Dougan Gordon 
Published: December 2012

ABSTRACT

MicroRNAs (miRNAs) are small noncoding molecules that control gene expression posttranscriptionally, with microRNA-155 (miR-155) one of the first to be implicated in immune regulation. Here, we show that miR-155-deficient mice are less able to eradicate a mucosal
Citrobacter rodentium
infection than wild-type C57BL/6 mice. miR-155-deficient mice exhibited prolonged colonization associated with a higher
C. rodentium
burden in gastrointestinal tissue and spread into systemic tissues. Germinal center formation and humoral immune responses against
C. rodentium
were severely impaired in infected miR-155-deficient mice. A similarly susceptible phenotype was observed in μMT mice reconstituted with miR-155-deficient B cells, indicating that miR-155 is required intrinsically for mediating protection against this predominantly luminal bacterial pathogen.
Molecular Pathways: Targeting Mdm2 and Mdm4 in Cancer Therapy
Authors: Li Qin , Lozano Guillermina 
Published: December 2012

Abstract
The p53 tumor suppressor is activated in response to cellular stresses to induce cell-cycle arrest, cellular senescence, and apoptosis. The p53 gene is inactivated by mutations in more than 50% of human tumors. In addition, tumor cells dampen p53 activities via overexpression of p53-negative regulators, in particular 2 structurally related proteins, Mdm2 and Mdm4. And yet, Mdm2 and Mdm4 possess p53-independent activities, which also contribute to tumor formation and progression. Given that Mdm2 and Mdm4 inhibit p53 activities to promote tumor development, small molecules and peptides were developed to abrogate the inhibition of p53 by Mdm proteins. Antitumor activities of these molecules have already been confirmed in preclinical studies and early-phase clinical trials. These research endeavors and clinical advances constitute the main focus of this review. Clin Cancer Res; 19(1); 34–41. ©2012 AACR.
Blocked Autophagy Using Lysosomotropic Agents Sensitizes Resistant Prostate Tumor Cells to the Novel Akt Inhibitor AZD5363
Authors: Lamoureux Francois , Thomas Christian , Crafter Claire , Kumano Masafumi , Zhang Fan , Davies Barry R. , Gleave Martin E. , Zoubeidi Amina 
Published: December 2012

Abstract
Purpose: Prostate cancer development is often associated with deletion or silencing of tumor suppressor phosphatase and tensin homolog (PTEN), a negative regulator of the phosphoinositide 3 kinase (PI3K)–Akt pathway, leading to resistance to various therapies in both the preclinical and clinical setting. Therefore, the PI3K–Akt pathway plays a central role in various cellular processes promoting survival signaling that can contribute to the malignant phenotype, and, consequently, is an attractive pharmacologic target. However, as single agents, the efficacy of AKT inhibitors may be limited by resistance mechanisms that result in minimal cell death in tumor cells.
Experimental Design: We investigated the effects of the Akt inhibitor AZD5363 on cell proliferation, cell cycle, apoptosis, and Akt downstream pathway proteins. Survival mechanisms induced by AZD5363 were investigated. We then examined the impacts of inhibition of autophagy in combination with AZD5363 on cell proliferation and apoptosis. Furthermore, the anticancer activity of combination treatment of the lysosomotropic inhibitor of autophagy (chloroquine) with the Akt inhibitor AZD5363 was evaluated in PC-3 prostate cancer xenografts.
Results: Here, we show that the Akt inhibitor AZD5363 affected the Akt downstream pathway by reducing p-mTOR, p-P70S6K, and p-S6K. While AZD5363 monotherapy induced G2 growth arrest and autophagy, it failed to induce significant apoptosis in PC-3 and DU145 prostate cancer cell lines. Blocking autophagy using pharmacologic inhibitors (3-methyladenine, chloroquine, and bafilomycin A) or genetic inhibitors (siRNA targeting Atg3 and Atg7) enhanced cell death induced by Akt inhibitor AZD5363 in these tumor prostate cell lines. Importantly, the combination of AZD5363 with chloroquine significantly reduced tumor volume by 84.9% compared with the control group and by 77.5% compared with either drug alone in PC3 xenografts.
Conclusion: Taken together, these data show that the Akt inhibitor AZD5363 synergizes with the lysosomotropic inhibitor of autophagy chloroquine to induce apoptosis and delay tumor progression in prostate cancer models that are resistant to monotherapy AZD5363, providing a new therapeutic approach potentially translatable to patients. Clin Cancer Res; 19(4); 833–44. ©2012 AACR.