bluuebunny/crossref_metadata_2025_split huggingface dataset:
New Insights into the Glycosylation of the Surface Layer Protein SgsE from
Geobacillus stearothermophilus
NRS 2004/3a
Authors: Steiner Kerstin , Pohlentz Gottfried , Dreisewerd Klaus , Berkenkamp Stefan , Messner Paul , Peter-Katalinić Jasna , Schäffer Christina 
Published: November 2006

ABSTRACT

The surface of
Geobacillus stearothermophilus
NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [→2)-α-
l
-Rha
p-
(1→3)-β-
l
-Rha
p
-(1→2)-α-L-Rha
p
-(1→] repeating units, with a 2-
O
-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known →3)-α-
l
-Rha
p-
(1→3)-α-
l
-Rha
p-
(1→ core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.
SalmonellaProduces an O-Antigen Capsule Regulated by AgfD and Important for Environmental Persistence
Authors: Gibson D. L. , White A. P. , Snyder S. D. , Martin S. , Heiss C. , Azadi P. , Surette M. , Kay W. W. 
Published: November 2006

ABSTRACTIn this study, we show thatSalmonellaproduces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. Structural analysis of purifiedSalmonellaextracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that ofSalmonella entericaserovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designatedyihU-yshAandyihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenicΔyihmutants, whereyihQandyihOwere shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates theyihoperons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since theyihgenes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.
The
Methanosarcina barkeri
Genome: Comparative Analysis with
Methanosarcina acetivorans
and
Methanosarcina mazei
Reveals Extensive Rearrangement within Methanosarcinal Genomes
Authors: Maeder Dennis L. , Anderson Iain , Brettin Thomas S. , Bruce David C. , Gilna Paul , Han Cliff S. , Lapidus Alla , Metcalf William W. , Saunders Elizabeth , Tapia Roxanne , Sowers Kevin R. 
Published: November 2006

ABSTRACT

We report here a comparative analysis of the genome sequence of
Methanosarcina barkeri
with those
of Methanosarcina acetivorans
and
Methanosarcina mazei
. The genome of
M. barkeri
is distinguished by having an organization that is well conserved with respect to the other
Methanosarcina
spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in
M. barkeri
, 746 had homologs with better than 80% identity to both
M. acetivorans
and
M. mazei
, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for
N
-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an
orc1
gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in
M. barkeri
. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large
M. acetivorans
genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the
M. barkeri
genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short
M. mazei
genome most closely approximates the putative ancestral organizational state of these species.
Structural Organization and Functional Properties of Miniature DNA Insertion Sequences in Yersiniae
Authors: De Gregorio Eliana , Silvestro Giustina , Venditti Rossella , Carlomagno Maria Stella , Di Nocera Pier Paolo 
Published: November 2006

ABSTRACTYPALs (Yersinia palindromic sequences) are miniature DNA insertions scattered along the chromosomes of yersiniae. The spread of these intergenic repeats likely occurred via transposition, as suggested by the presence of target site duplications at their termini and the identification of syntenic chromosomal regions which differ in the presence/absence of YPAL DNA amongYersiniastrains. YPALs tend to be inserted closely downstream from the stop codon of flanking genes, and many YPAL targets overlap rho-independent transcriptional terminator-like sequences. This peculiar pattern of insertion supports the hypothesis that most of these repeats are cotranscribed with upstream sequences into mRNAs. YPAL RNAs fold into stable hairpins which may modulate mRNA decay. Accordingly, we found that YPAL-positive transcripts accumulate inYersinia enterocoliticacells at significantly higher levels than homologous transcripts lacking YPAL sequences in their 3′ untranslated region.
Cooperative and Critical Roles for Both G Domains in the GTPase Activity and Cellular Function of Ribosome-Associated
Escherichia coli
EngA
Authors: Bharat Amrita , Jiang Mengxi , Sullivan Susan M. , Maddock Janine R. , Brown Eric D. 
Published: November 2006

ABSTRACT

To probe the cellular phenotype and biochemical function associated with the G domains of
Escherichia coli
EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an
engA
conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His
6
-EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.
Heme Utilization in
Campylobacter jejuni
Authors: Ridley Kristian A. , Rock Jonathan D. , Li Ying , Ketley Julian M. 
Published: November 2006

ABSTRACT

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes
chuABCD
and a putative heme oxygenase gene (Cj1613c), has been identified in
Campylobacter jejuni
NCTC 11168. Mutation of
chuA
or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of
chuB
,
-C
, or
-D
only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (
A
TP-
b
inding
c
assette) transport system(s) for heme is present in
C. jejuni
. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess
chuC
, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-
chuA
intergenic spacer region revealed
chuABCD
and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur
Cj
in gel retardation studies. Absorbance spectroscopy of purified recombinant His
6
-Cj1613c revealed a 1:1 heme:His
6
-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in
C. jejuni.
Design of a Seven-GenomeEscherichia coliMicroarray for Comparative Genomic Profiling
Authors: Willenbrock Hanni , Petersen Anne , Sekse Camilla , Kiil Kristoffer , Wasteson Yngvild , Ussery David W. 
Published: November 2006

ABSTRACTWe describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequencedEscherichia coligenomes in addition to several sequencedE. coliplasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Δstx2::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its φ3538 (Δstx2::cat) phage element originated from theE. coli3538 (Δstx2::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer φ3538 (Δstx2::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis.
A Conserved Tetranucleotide Repeat Is Necessary for Wild-Type Expression of the
Moraxella catarrhalis
UspA2 Protein
Authors: Attia Ahmed S. , Hansen Eric J. 
Published: November 2006

ABSTRACT

The UspA2 protein has been shown to be directly involved in the serum-resistant phenotype of
Moraxella catarrhalis
. The predicted 5′-untranslated regions (UTR) of the
uspA2
genes in several different
M. catarrhalis
strains were shown to contain various numbers (i.e., 6 to 23) of a heteropolymeric tetranucleotide (AGAT) repeat. Deletion of the AGAT repeats from the
uspA2
genes in the serum-resistant
M. catarrhalis
strains O35E and O12E resulted in a drastic reduction in UspA2 protein expression and serum resistance. PCR and transformation were used to construct a series of
M. catarrhalis
O12E strains that differed only in the number of AGAT repeats in their
uspA2
genes. Expression of UspA2 was maximal in the presence of 18 AGAT repeats, although serum resistance attained wild-type levels in the presence of as few as nine AGAT repeats. Increased UspA2 expression was correlated with both increased binding of vitronectin and decreased binding of polymerized C9. Real-time reverse transcription-PCR analysis showed that changes in the number of AGAT repeats affected the levels of
uspA2
mRNA, with 15 to 18 AGAT repeats yielding maximal levels. Primer extension analysis indicated that these AGAT repeats were contained in the 5′-UTR of the
uspA2
gene. The mRNA transcribed from a
uspA2
gene containing 18 AGAT repeats was found to have a longer half-life than that transcribed from a
uspA2
gene lacking AGAT repeats. These data confirm that the presence of the AGAT repeats in the 5′-UTR of the
uspA2
gene is necessary for both normal expression of the UspA2 protein and serum resistance.
Epidermal growth factor receptor status in anaplastic thyroid carcinoma
Authors: Lee Dae Ho , Lee Geon Kook , Kong Sun-young , Kook Myoung Chul , Yang Sun Kyung , Park So Yeon , Park Seong Hoe , Keam Bhumsuk , Park Do Joon , Cho Bo Youn , Kim Seok Won , Chung Ki-Wook , Lee Eun Sook , Kim Sun Wook 
Published: November 2006

Background:The epidermal growth factor receptor (EGFR) has been reported to be overexpressed in anaplastic thyroid carcinoma (ATC). In vitro studies have shown that EGFR tyrosine kinase inhibitors (TKIs) greatly inhibit cellular growth and induced apoptosis in the ATC cell lines, while somatic mutations in the tyrosine kinase domain or an increased gene copy number are associated with increased sensitivity to TKIs in non-small cell lung cancer.Aim:To investigate the prevalence of EGFR overexpression, gene amplification and activating mutation in the tyrosine kinase domain in patients with ATC.Methods:TheEGFRgene status and protein expression were investigated by direct DNA sequencing of the hot-spot regions in exons 18, 19 and 21, fluorescence in situ hybridisation (FISH), and immunohistochemistry in tumour tissues from 23 patients with ATC.Results:On mutational analysis and FISH, neither mutations in the hot-spots nor gene amplification was observed. However, high polysomy was identified in 14/23 (60.9%) patients with ATC. All cases with immunohistochemistry (IHC) positivity (n = 6) had high polysomy, whereas 8/17 (47.1%) cases with IHC negativity had high polysomy (p = 0.048). High polysomy was observed in all 10 cases with giant cell subtype, but in only 4/11 (36.3%) with squamoid and 0/2 with spindle cell sarcomatoid subtype. There was no statistically significant correlation between FISH positivity of ATC tumour and presence of well-differentiated component.Conclusion:Despite the low incidence of somaticEGFRgene mutation and amplification in the study samples, in view of the fact that high polysomy was often identified by FISH, as well as the current lack of therapeutic options, EGFR TKIs are worth investigating for treating the patients with ATC who have at least giant cell subtype.
A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability
Authors: Kim Grace J. , Fiskum Gary M. , Morgan William F. 
Published: November 2006

Abstract
Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones. (Cancer Res 2006; 66(21): 10377-83)
Activating Transcription Factor 2 Mediates Matrix Metalloproteinase-2 Transcriptional Activation Induced by p38 in Breast Epithelial Cells
Authors: Song Hyun , Ki Sung Hwan , Kim Sang Geon , Moon Aree 
Published: November 2006

Abstract
Mounting evidence suggests a role for matrix metalloproteinase (MMP)-2 in the malignant progression of breast cancer cells. We showed previously that H-Ras, but not N-Ras, induced invasion of MCF10A human breast epithelial cells through Rac-MKK3/6-p38 pathway resulted in MMP-2 up-regulation. Activation of p38 pathway by MKK6 caused a selective up-regulation of MMP-2. In this study, we aimed to elucidate the transcriptional regulation of MMP-2 by p38 pathway leading to the invasive phenotype of MCF10A cells. By using 5′ deletion mutant constructs of MMP-2 promoter, we showed that deletion of the region containing activator protein-1 (AP-1) site caused the greatest reduction of MMP-2 promoter activity both in MKK6- and H-Ras-activated MCF10A cells, suggesting that the AP-1 binding site is critical for the MMP-2 promoter activation. DNA binding and transcriptional activities of AP-1 were increased by MKK6 or H-Ras as evidenced by electrophoretic mobility shift assay and luciferase assay using an AP-1-driven plasmid. By doing immunoinhibition assay and chromatin immunoprecipitation assay, we revealed the activating transcription factor (ATF) 2 as a transcription factor for MMP-2 gene expression through binding to the functional AP-1 site. Activation of ATF2, which depended on p38 activity, was crucial for MMP-2 promoter activity as well as induction of invasive and migrative phenotypes in MCF10A cells. This is the first report revealing ATF2 as an essential transcription factor linking MKK3/6-p38 signaling pathway to MMP-2 up-regulation, providing evidence for a direct role of ATF2 activation in malignant phenotypic changes of human breast epithelial cells. (Cancer Res 2006; 66(21): 10487-96)
p16INK4a Modulates p53 in Primary Human Mammary Epithelial Cells
Authors: Zhang Jianmin , Pickering Curtis R. , Holst Charles R. , Gauthier Mona L. , Tlsty Thea D. 
Published: November 2006

Abstract
p16INK4a (p16) and p53 are tumor suppressor genes that are inactivated during carcinogenesis in many tumors. Here we show that p16 gene activity inversely modulates p53 status and function in primary human mammary epithelial cells. Reduced levels of p16 protein stabilize p53 protein through inhibition of proteolytic degradation, and this increase in p53 protein levels enhances the cellular response to radiation, represses proliferation, and transcriptionally activates downstream targets. Stabilization of p53 is mediated through the retinoblastoma/E2F/p14ARF/murine double minute-2 pathway. However, we have observed that p16 does not modulate p53 in fibroblasts, indicating a possible cell type–specific regulation of this pathway. (Cancer Res 2006; 66(21): 10325-31)
Alterations of the Tumor Suppressor Gene ARLTS1 in Ovarian Cancer
Authors: Petrocca Fabio , Iliopoulos Dimitrios , Qin Haiyan R. , Nicoloso Milena S. , Yendamuri Say , Wojcik Sylwia E. , Shimizu Masayoshi , Di Leva Gianpiero , Vecchione Andrea , Trapasso Francesco , Godwin Andrew K. , Negrini Massimo , Calin George A. , Croce Carlo M. 
Published: November 2006

Abstract
ARLTS1 is a tumor suppressor gene initially described as a low-penetrance cancer gene: a truncated Trp149Stop (MUT) polymorphism is associated with general familial cancer aggregation and, particularly, high-risk familial breast cancer. DNA hypermethylation has been identified as a mechanism of ARLTS1 expression down-regulation in lung carcinomas and B-cell chronic lymphocytic leukemia. We found that, in the majority of ovarian carcinomas (61.5%) and in a significant proportion of ovarian and breast cancer cell lines (45%), ARLTS1 is strongly down-regulated due to DNA methylation in its promoter region. After ARLTS1 restoration by adenoviral transduction, only the negative TOV-112 and the homozygously mutated (MUT) MCF7 cells, but not the OV-90 cells expressing a normal ARLTS1 product, underwent apoptosis and inhibition of cell growth. Furthermore, ARLTS1 reexpression significantly reduced the tumorigenic potential of TOV-112 in nude mice. On the contrary, the ARLTS1-MUT induced significantly lower levels of apoptosis in infected cells and reduced in vivo tumorigenesis only partially, supporting the hypothesis that Trp149Stop polymorphism is retained in the general population and predisposes to cancer because of a reduction, but not full loss, of normal ARLTS1 function. (Cancer Res 2006; 66(21): 10287-91)