bluuebunny/crossref_metadata_2025 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

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

In 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

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

YPALs (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

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

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

We 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

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.
GaSb/AlGaSb COMPOUND SEMICONDUCTORS GROWN BY MOCVD FOR OPTOELECTRONIC APPLICATIONS
Authors: RAMELAN A. H. , YAHYA I. , PRASODJO  , GOLDYS E. M.
Published: November 2006

Al x Ga 1-x Sb films in the regime 0 ≤ x ≤ 0.30 have been grown by metalorganic chemical vapor deposition on GaAs and GaSb substrates using TMAl , TMGa and TMSb precursors. We report the effects of growth conditions on the optical properties. Samples grown at temperatures of 540°C, 580°C and 600°C and a V/III ratio of 1 have been investigated. The Al x Ga 1-x Sb layers grown at 580°C and 600°C with a V/III ratio of 1 and Al content in the range of 0.5% to 25% were found to exhibit excellent optical quality with a very high optical transmission at energies below the bandgap. The principle photoluminescence features observed are attributed to bound exciton and donor-acceptor transitions with FWHM comparable to the best values reported elsewhere.
DOUBLE PASS S-BAND EDFA
Authors: AHMAD HARITH , HARUN SULAIMAN WADI
Published: November 2006

A double-pass erbium-doped fiber amplifier (EDFA) based on a depressed cladding erbium-doped fiber, which operates in the short wavelength (S-band) region is proposed and demonstrated. This amplifier provides a small signal gain as high as 30 dB at 1500 nm, using only 15 m depressed cladding erbium-doped fiber (EDF) and 130 mW of 980 nm pump power. Compared to the single-pass configuration, this amplifier shows a gain enhancement of about 14 dB for pump powers above 120 mW. However, noise figures penalties of less than 0.9 dB are obtained for these pump powers. The proposed double-pass amplifier will play an important role in the development of a practical S-band EDFA from the perspective of economical usage of EDF and pump power.
BLOCH AMPLITUDES AND ENERGY OF SLOW LIGHT IN A TAPERED GRATING
Authors: VALKERING THEO P.
Published: November 2006

Bloch amplitudes and energy density for traveling and standing waves in an apodized grating at frequencies just outside the band gap are investigated. On the basis of exact coupled mode equations for the local Bloch amplitudes, based on the transfer matrix of the grating units, the concept of local reflectance is developed. This concept appears to be particularly useful to find upper and lower bounds on amplitudes and energy density. Analytical results are tested numerically for a polynomial apodization profile.
Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes
Authors: Leduc-Nadeau Alexandre , Lahjouji Karim , Bissonnette Pierre , Lapointe Jean-Yves , Bichet Daniel G.
Published: November 2006

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na+-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.
Encoding of Movement Dynamics by Purkinje Cell Simple Spike Activity During Fast Arm Movements Under Resistive and Assistive Force Fields
Authors: Yamamoto Kenji , Kawato Mitsuo , Kotosaka Shinya , Kitazawa Shigeru
Published: November 2006

It is controversial whether simple-spike activity of cerebellar Purkinje cells during arm movements encodes movement kinematics like velocity or dynamics like muscle activities. To examine this issue, we trained monkeys to flex or extend the elbow by 45° in 400 ms under resistive and assistive force fields but without altering kinematics. During the task movements after training, simple-spike discharges were recorded in the intermediate part of the cerebellum in lobules V–VI, and electromyographic activity was recorded from arm muscles. Velocity profiles (kinematics) in the two force fields were almost identical to each other, whereas not only the electromyographic activities (dynamics) but also simple-spike activities in many Purkinje cells differed distinctly depending on the type of force field. Simple-spike activities encoded much larger mutual information with the type of force field than that with the residual small difference in the height of peak velocity. The difference in simple-spike activities averaged over the recorded Purkinje-cells increased ∼40 ms before the appearance of the difference in electromyographic activities between the two force fields, suggesting that the difference of simple-spike activities could be the origin of the difference of muscle activities. Simple-spike activity of many Purkinje cells correlated with electromyographic activity with a lead of ∼80 ms, and these neurons had little overlap with another group of neurons the simple-spike activity of which correlated with velocity profiles. These results show that simple-spike activity of at least a group of Purkinje cells in the intermediate part of cerebellar lobules V–VI encodes movement dynamics.
Neural Rate and Timing Cues for Detection and Discrimination of Amplitude-Modulated Tones in the Awake Rabbit Inferior Colliculus
Authors: Nelson Paul C. , Carney Laurel H.
Published: November 2006

Neural responses to amplitude-modulated (AM) tones in the unanesthetized rabbit inferior colliculus (IC) were studied in an effort to establish explicit relationships between physiological and psychophysical measures of temporal envelope processing. Specifically, responses to variations in modulation depth ( m) at the cell’s best modulation frequency, with and without modulation maskers, were quantified in terms of average rate and synchronization to the envelope over the entire perceptual dynamic range of depths. Statistically significant variations in the metrics were used to define neural AM detection and discrimination thresholds. Synchrony emerged at modulation depths comparable with psychophysical AM detection sensitivities in some neurons, whereas the lowest rate-based neural thresholds could not account for psychoacoustical thresholds. The majority of rate thresholds (85%) were −10 dB or higher (in 20 log m), and 16% of the population exhibited no systematic dependence of average rate on m. Neural thresholds for AM detection did not decrease systematically at higher SPLs (as observed psychophysically): thresholds remained constant or increased with level for most cells tested at multiple sound-pressure levels (SPLs). At depths higher than the rate-based detection threshold, some rate modulation-depth functions were sufficiently steep with respect to the across-trial variability of the rate to predict depth discrimination thresholds as low as 1 dB (comparable with the psychophysics). Synchrony, on the other hand, did not vary systematically with m in many cells at high modulation depths. A simple computational model was extended to reproduce several features of the modulation frequency and depth dependence of both transient and sustained pure-tone responders.
Modeling, description, and characterization of fractal pore via mathematical morphology
Authors: Teo Lay Lian , Sagar B. S. Daya
Published: November 2006

The aim of this paper is to provide description of fast, simple computational algorithms based upon mathematical morphology techniques to extract descriptions of pore channels—throats—and bodies and to represent them in 3D space, and to produce statistical characterization of their descriptions. Towards this goal, a model fractal binary pore is considered and is eroded recursively to generate different slices possessing decreasing degrees of porosity. By employing simple morphology‐based approach, each slice of this pore space is decomposed into pore‐channel, pore‐throat, and pore‐body, which are abstract structures that summarize the overall connectivity, orientation, and shape of the pore space. We consider the pore slices and their corresponding morphological quantities to stack them to further represent them in 3D space. We further provide a formulation essentially based on set theory to represent these three morphologic quantities to connect them appropriately across slices. The connected quantities are further fragmented to designate each fragmented portion with orders ranging from 1 to N.
Dynamics of flexible shells and Sharkovskiy′s periodicity
Authors: Krysko Vadim A. , Awrejcewicz Jan , Saveleva Natalya E. , Krysko Anton V.
Published: November 2006

Complex vibration of flexible elastic shells subjected to transversal and sign‐changeable local load in the frame of nonlinear classical theory is studied. A transition from partial to ordinary differential equations is carried out using the higher‐order Bubnov‐Galerkin approach. Numerical analysis is performed applying theoretical background of nonlinear dynamics and qualitative theory of differential equations. Mainly the so‐called Sharkovskiy periodicity is studied.
A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability
Authors: Kim Grace J. , Fiskum Gary M. , Morgan William F.
Published: November 2006

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

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

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

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)
Targeting Cancer Cells by Novel Engineered Modular Transporters
Authors: Gilyazova Dinara G. , Rosenkranz Andrey A. , Gulak Pavel V. , Lunin Vladimir G. , Sergienko Olga V. , Khramtsov Yuri V. , Timofeyev Kirill N. , Grin Mikhail A. , Mironov Andrey F. , Rubin Andrey B. , Georgiev Georgii P. , Sobolev Alexander S.
Published: November 2006

A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and α/β-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and ·OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications. (Cancer Res 2006; 66(21): 10534-40)
Inappropriate Activation of the Androgen Receptor by Nonsteroids: Involvement of the Src Kinase Pathway and Its Therapeutic Implications
Authors: Desai Sonal J. , Ma Ai-Hong , Tepper Clifford G. , Chen Hong-Wu , Kung Hsing-Jien
Published: November 2006

The inappropriate activation of androgen receptor (AR) by nonsteroids is considered a potential mechanism in the emergence of hormone-refractory prostate tumors, but little is known about the properties of these “pseudoactivated” AR. Here, we present the first comprehensive analysis closely examining the properties of AR activated by the neuropeptide bombesin that distinguish it from androgen-activated AR. We show that bombesin-activated AR (a) is required for bombesin-induced growth of LNCaP cells, (b) has a transcriptional profile overlapping with, but not identical to, androgen-activated AR, (c) activates prostate-specific antigen by preferentially binding to its proximal promoter, and (d) assembles a distinct coactivator complex. Significantly, we found that Src kinase is critical for bombesin-induced AR-mediated activity and is required for translocation and transactivation of AR. Additionally, we identify c-Myc, a Src target gene, to be activated by bombesin and a potential coactivator of AR-mediated activity specific to bombesin-induced signaling. Because Src kinase is often activated by other nonsteroids, such as other neuropeptides, growth factors, chemokines, and cytokines, our findings have general applicability and provide rationale for investigating the efficacy of the Src kinase pathway as a target for the prevention of relapsed prostate cancers. (Cancer Res 2006; 66(21): 10449-59)
Linkage Disequilibrium and Haplotype Homozygosity in Population Samples Genotyped at a High Marker Density
Authors: Wang Hui , Lin Chia-Ho , Service Susan , Chen Yuguo , Freimer Nelson , Sabatti Chiara
Published: November 2006

Objective: Analyze the information contained in homozygous haplotypes detected with high density genotyping. Methods: We analyze the genotypes of ∼2,500 markers on chr 22 in 12 population samples, each including 200 individuals. We develop a measure of disequilibrium based on haplotype homozygosity and an algorithm to identify genomic segments characterized by non-random homozygosity (NRH), taking into account allele frequencies, missing data, genotyping error, and linkage disequilibrium. Results: We show how our measure of linkage disequilibrium based on homozygosity leads to results comparable to those of R2, as well as the importance of correcting for small sample variation when evaluating D′. We observe that the regions that harbor NRH segments tend to be consistent across populations, are gene rich, and are characterized by lower recombination. Conclusions: It is crucial to take into account LD patterns when interpreting long stretches of homozygous markers.
Gamma Knife Radiosurgery in the Treatment of Choroidal Neovascularization (Wet-Type Macular Degeneration)
Authors: Henderson Mark A. , Valluri Shailaja , Lo Simon S. , Witt Thomas C. , Worth Robert M. , Danis Ronald P. , Timmerman Robert D.
Published: November 2006

We evaluated retrospectively our institutional experience in the treatment of macular degeneration with gamma knife radiosurgery (GKR). Treatment was delivered in a single shot of 12 Gy. Seven patients were treated between March of 1999 and May of 2000. The median duration of follow-up was 2.2 years. The majority of patients maintained stable visual acuity after treatment. Our series indicates that GKR may be useful as a salvage treatment for patients who have failed or are ineligible for other treatments for their macular degeneration. Further studies are needed to better define the role of GKR in the treatment of macular degeneration.
Interleukin-6 Genetic Ablation Protects from Trinitrobenzene Sulfonic Acid-Induced Colitis in Mice
Authors: Gay Jérôme , Kokkotou Efi , O’Brien Michael , Pothoulakis Charalabos , Karalis Katia P.
Published: November 2006

Background: Interleukin (IL)-6 is a proinflammatory cytokine implicated in the pathogenesis of inflammatory bowel disease. IL-6 is locally upregulated in inflammatory bowel disease patients and in murine models of experimental colitis. Treatment with anti-IL-6 receptor antibody ameliorates intestinal inflammation. Objective: It was the aim of this study to investigate the role of genetic IL-6 deficiency in trinitrobenzene sulfonic acid (TNBS)-mediated colitis, an experimental model inflammation that shares several features with Crohn’s disease in humans. Methods: Acute colitis was induced in wild-type and IL-6-deficient (Il-6–/–) mice by intracolonic administration of TNBS. Forty-eight hours after treatment, the local and systemic features of inflammation, i.e. food intake, weight loss, histological markers of colitis, cytokine expression by real-time PCR, food intake and body weight changes, were assessed. Results: In wild-type mice, TNBS administration increased both IL-6 serum levels and local expression of IL-6 by 36 and 9 fold, respectively, compared with control, vehicle-injected mice. Compared with the wild-type mice, the Il-6–/– mice had significantly reduced intestinal inflammation as evidenced by epithelial damage, neutrophil infiltration, colon thickness and proinflammatory cytokine expression, following treatment with TNBS. Moreover, baseline levels of the antiinflammatory cytokines IL-10 and tumor growth factor-β were significantly elevated in Il-6–/–compared with the wild-type mice. Conclusions: Our results demonstrate that Il-6–/–are partially protected from the development of TNBS-induced acute experimental colitis, most likely via their significantly elevated baseline levels of antiinflammatory cytokines.
Intracranial Pressure Parameters in Idiopathic Normal Pressure Hydrocephalus Patients with or without Improvement of Cognitive Function after Shunt Treatment
Authors: Foss Tonje , Eide Per Kristian , Finset Arnstein
Published: November 2006

Background/Aims: Previous research has shown improvements in the clinical triad (gait, incontinence and cognitive dysfunction) after shunt surgery in idiopathic normal pressure hydrocephalus (iNPH) patients with intracranial pulse pressure amplitudes >4–5 mm Hg, the pulse amplitudes being <4 mm Hg in the nonresponders. However, it is unknown whether similar differences exist regarding change in cognitive functioning after shunt surgery. The aim of this study was to compare preoperative intracranial pressure (ICP) parameters between iNPH that either improved or not improved in cognitive function after shunt treatment. Methods: Neuropsychological testing was performed before and after surgery in 27 consecutive iNPH patients treated with ventriculoperitoneal shunts. The ICP recordings were performed as part of routine preoperative assessment, stored as raw data files, and analyzed retrospectively. The mean ICP as well as single ICP wave amplitudes were computed and analyzed in consecutive 6-second time windows. Results: Significant improvement in neuropsychological tests, defined as a 4-point improvement in Folstein Mini-Mental State Examination or improvement by one standard deviation in 50% of subtests of Dementia Rating Scale, was found in 12 patients (44%; shunt responders). In these patients, mean ICP was similar though the mean ICP wave amplitude was significantly higher than in the shunt nonresponders. Conclusions: While preoperative mean ICP was similar, the mean ICP wave amplitudes were considerably higher in iNPH patients with significant change in cognitive function following shunt surgery.
Immune regulation by pretenders: cell-to-cell transfers of HLA-G make effector T cells act as regulatory cells
Authors: LeMaoult Joel , Caumartin Julien , Daouya Marina , Favier Benoit , Rond Solene Le , Gonzalez Alvaro , Carosella Edgardo D.
Published: November 2006

Trogocytosis is the uptake of membrane fragments from one cell by another and has been described for immune cells in mice and humans. Functional consequences of trogocytosis are emerging, but a dramatic immune function has still to be associated with it. Here we show that some resting, and most activated, CD4+ and CD8+ T cells acquire immunosuppressive HLA-G1 from antigen-presenting cells (APCs) in a few minutes. Acquisition of HLA-G through membrane transfers does not change the real nature of the T cells but immediately reverses their function from effectors to regulatory cells. These regulatory cells can inhibit allo-proliferative responses through HLA-G1 that they acquired. These data demonstrate that trogocytosis of HLA-G1 leads to instant generation of a new type of regulatory cells, which act through cell-surface molecules they temporarily display but do not express themselves. Such regulatory cells whose existence is most likely limited in space and time might constitute an “emergency” immune suppression mechanism used by HLA-G–expressing tissues to protect themselves against immune aggression. In addition, T cells acquire from HLA-G–expressing APCs their HLA-G–dependent capability to induce the slower differentiation of regulatory cells that act independently of HLA-G. These data re-emphasize the significance of HLA-G expression in normal and pathologic situations.
FGF2 posttranscriptionally down-regulates expression of SDF1 in bone marrow stromal cells through FGFR1 IIIc
Authors: Nakayama Takayuki , Mutsuga Noriko , Tosato Giovanna
Published: November 2006

The chemokine stromal cell–derived factor-1 (SDF-1) is constitutively expressed by bone marrow stromal cells and plays key roles in hematopoiesis. Fibroblast growth factor 2 (FGF2), a member of the FGF family that plays important roles in developmental morphogenic processes, is abnormally elevated in the bone marrow from patients with clonal myeloid disorders and other disorders where normal hematopoiesis is impaired. Here, we report that FGF2 reduces SDF-1 secretion and protein content in bone marrow stromal cells. By inhibiting SDF-1 production, FGF2 compromises stromal cell support of hematopoietic progenitor cells. Reverse-transcriptase–polymerase chain reaction (RT-PCR) analysis revealed that bone marrow stromal cells express 5 FGF receptors (FGFRs) among the 7 known FGFR subtypes. Blocking experiments identified FGFR1 IIIc as the receptor mediating FGF2 inhibition of SDF-1 expression in bone marrow stromal cells. Analysis of the mechanisms underlying FGF2 inhibition of SDF-1 production in bone marrow stromal cells revealed that FGF2 reduces the SDF-1 mRNA content by posttranscriptionally accelerating SDF-1 mRNA decay. Thus, we identify FGF2 as an inhibitor of SDF-1 production in bone marrow stromal cells and a regulator of stromal cell supportive functions for hematopoietic progenitor cells.
Bone morphogenic protein antagonist Drm/gremlin is a novel proangiogenic factor
Authors: Stabile Helena , Mitola Stefania , Moroni Emanuela , Belleri Mirella , Nicoli Stefania , Coltrini Daniela , Peri Francesco , Pessi Antonello , Orsatti Laura , Talamo Fabio , Castronovo Vincent , Waltregny David , Cotelli Franco , Ribatti Domenico , Presta Marco
Published: November 2006

Angiogenesis plays a key role in various physiologic and pathologic conditions, including tumor growth. Drm/gremlin, a member the Dan family of bone morphogenic protein (BMP) antagonists, is commonly thought to affect different processes during growth, differentiation, and development by heterodimerizing various BMPs. Here, we identify Drm/gremlin as a novel proangiogenic factor expressed by endothelium. Indeed, Drm/gremlin was purified to homogeneity from the conditioned medium of transformed endothelial cells using an endothelial-cell sprouting assay to follow protein isolation. Accordingly, recombinant Drm/gremlin stimulates endothelial-cell migration and invasion in fibrin and collagen gels, binds with high affinity to various endothelial cell types, and triggers tyrosine phosphorylation of intracellular signaling proteins. Also, Drm/gremlin induces neovascularization in the chick embryo chorioallantoic membrane. BMP4 does not affect Drm/gremlin interaction with endothelium, and both molecules exert a proangiogenic activity in vitro and in vivo when administered alone or in combination. Finally, Drm/gremlin is produced by the stroma of human tumor xenografts in nude mice, and it is highly expressed in endothelial cells of human lung tumor vasculature when compared with non-neoplastic lung. Our observations point to a novel, previously unrecognized capacity of Drm/gremlin to interact directly with target endothelial cells and to modulate angiogenesis.
Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects
Authors: Wang Jikui , Nagy Andre , Larsson Jonas , Dudas Marek , Sucov Henry M , Kaartinen Vesa
Published: November 2006

Background

Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood.

Results

We deleted the TGF-β type I receptor Alk5 specifically in the mouse neural crest cell lineage. Failure in signaling via ALK5 leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. While ALK5 is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells.

Conclusion

Our results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development.