bluuebunny/crossref_metadata_2025_split huggingface dataset:
Global Analysis of Proteins Synthesized by
Mycobacterium smegmatis
Provides Direct Evidence for Physiological Heterogeneity in Stationary-Phase Cultures
Authors: Blokpoel Marian C. J. , Smeulders Marjan J. , Hubbard Julia A. M. , Keer Jacquie , Williams Huw D. 
Published: September 2005

ABSTRACT

We have characterized the induction kinetics of ∼1,700 proteins during entry into and survival in carbon-starved stationary phase by
Mycobacterium smegmatis
. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in ≥128-day stationary-phase cultures (Spr
128↑
proteins). A number of Spr
128↑
proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr
128↑
protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the
rpsL
gene promoter (which heads the
Mycobacterium smegmatis
operon encoding the
tuf
gene encoding EF-Tu) or the
rrnA
promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so
rpsL
expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing
rpsL
at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.
Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box
Authors: Hands Sarah L. , Holland Lisa E. , Vankemmelbeke Mireille , Fraser Lauren , Macdonald Colin J. , Moore Geoffrey R. , James Richard , Penfold Christopher N. 
Published: September 2005

ABSTRACT

The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill
Escherichia coli
cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.
Role of RcsF in Signaling to the Rcs Phosphorelay Pathway inEscherichia coli
Authors: Majdalani Nadim , Heck Michael , Stout Valerie , Gottesman Susan 
Published: September 2005

ABSTRACTThercsphosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of theyojNgene, now renamedrcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both therprAandcpspromoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting this signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of anrcsCallele,rcsC137, that leads to high-level constitutive expression of bothcpsandrprAsuggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.
Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile,
Thermus thermophilus
: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance
Authors: Miyazaki Junichi , Asada Kuniko , Fushinobu Shinya , Kuzuyama Tomohisa , Nishiyama Makoto 
Published: September 2005

ABSTRACT

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from
Thermus thermophilus
(TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related β-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.
A Metabolic Enzyme as a Primary Virulence Factor ofMycoplasma mycoidessubsp.mycoidesSmall Colony
Authors: Pilo Paola , Vilei Edy M. , Peterhans Ernst , Bonvin-Klotz Laetitia , Stoffel Michael H. , Dobbelaere Dirk , Frey Joachim 
Published: September 2005

ABSTRACTDuring evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast,Mycoplasmaspecies, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenicMycoplasmaspecies lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenicMycoplasmaspecies cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant inMycoplasma mycoidessubsp.mycoidesSmall Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2formed byl-α-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected withM. mycoidessubsp.mycoidesSC in the presence of physiological amounts of glycerol, H2O2is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.
Novel
nirK
Cluster Genes in
Nitrosomonas europaea
Are Required for NirK-Dependent Tolerance to Nitrite
Authors: Beaumont Hubertus J. E. , Lens Sylvia I. , Westerhoff Hans V. , van Spanning Rob J. M. 
Published: September 2005

ABSTRACT

Nitrite reductase (NirK) of
Nitrosomonas europaea
confers tolerance to nitrite (NO
2
−
). The
nirK
gene is clustered with three genes of unknown physiological function:
ncgABC
. At present, this organization is unique to nitrifying bacteria. Here we report that the
ncgABC
gene products facilitate NirK-dependent NO
2
−
tolerance by reversing the negative physiological effect that is associated with the activity of NirK in their absence. We hypothesize that the
ncg
gene products are involved in the detoxification of nitric oxide that is produced by NirK.
Regulation of
sigL
Expression by the Catabolite Control Protein CcpA Involves a Roadblock Mechanism in
Bacillus subtilis
: Potential Connection between Carbon and Nitrogen Metabolism
Authors: Choi Soo-Keun , Saier Milton H. 
Published: September 2005

ABSTRACT

A catabolite-responsive element (CRE), a binding site for the CcpA transcription factor, was identified within the
sigL
structural gene encoding σ
L
in
Bacillus subtilis
. We show that CcpA binds to this CRE to regulate
sigL
expression by a “roadblock” mechanism and that this mechanism in part accounts for catabolite repression of σ
L
-directed
levD
operon expression.
Gefitinib (“Iressa”, ZD1839), an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Reverses Breast Cancer Resistance Protein/ABCG2–Mediated Drug Resistance
Authors: Nakamura Yoichi , Oka Mikio , Soda Hiroshi , Shiozawa Ken , Yoshikawa Megumi , Itoh Akiko , Ikegami Yoji , Tsurutani Junji , Nakatomi Katsumi , Kitazaki Takeshi , Doi Seiji , Yoshida Hisahiro , Kohno Shigeru 
Published: September 2005

Abstract
Gefitinib (“Iressa”, ZD1839) is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, and the single agent is clinically effective in non–small cell lung cancer. Although gefitinib combined with various cytotoxic agents has been reported to enhance cytotoxicity in vitro and in mouse models, the mechanism remains undetermined. Here, to explore the mechanism with topoisomerase I inhibitors, we focused on the efflux pump of the breast cancer resistance protein (BCRP/ABCG2), and then examined whether gefitinib restored drug sensitivity in multidrug-resistant cancer cells overexpressing BCRP. We used PC-6 human small cell lung cancer cells and multidrug-resistant PC-6/SN2-5H cells selected with SN-38 of the active metabolite of irinotecan, and BCRP-overexpressing MCF-7/MX cells selected with mitoxantrone and BCRP cDNA transfectant MCF-7/clone 8 cells. Drug sensitivity against anticancer drugs was determined by tetrazolium dye assay, and intracellular topotecan accumulation by FACScan. The topotecan transport study was done using the plasma membrane vesicles of PC-6/SN2-5H cells. The resistant PC-6/SN2-5H cells overexpressed BCRP but not epidermal growth factor receptor mRNA. Ten micromoles of gefitinib reversed topotecan, SN-38, and mitoxantrone resistance, and increased the intracellular topotecan accumulation in the resistant cells but not in the parental cells. Furthermore, gefitinib inhibited the topotecan transport into the vesicles, and the Ki value was 1.01 ± 0.09 μmol/L in the Dixon plot analysis, indicating direct inhibition of BCRP by gefitinib. However, gefitinib was not transported into the vesicles with the high-performance liquid chromatography method. These results indicate that gefitinib reverses BCRP-mediated drug resistance by direct inhibition other than competitive inhibition as a BCRP substrate. Combination of gefitinib and topoisomerase I inhibitors could be clinically effective in cancers expressing BCRP.
Tid-1 Interacts with the von Hippel-Lindau Protein and Modulates Angiogenesis by Destabilization of HIF-1α
Authors: Bae Moon-Kyoung , Jeong Joo-Won , Kim Se-Hee , Kim Soo-Young , Kang Hye Jin , Kim Dong-Min , Bae Soo-Kyung , Yun Il , Trentin Grace A. , Rozakis-Adcock Maria , Kim Kyu-Won 
Published: September 2005

Abstract
The von Hippel-Lindau protein (pVHL) is a major tumor suppressor protein and also associated with the inhibition of angiogenesis via HIF-1α ubiquitination and proteasomal degradation. To further elucidate the biological activity of pVHL in angiogenesis, pVHL-interacting proteins were screened using the yeast two-hybrid system. We found that a mouse homologue of the long form of Drosophila tumor suppressor l(2)tid, Tid-1L, directly interacts with pVHL in vitro and in vivo. Furthermore, Tid-1L protein; enhanced the interaction between HIF-1α and pVHL, leading to the destabilization of HIF-1α protein; therefore, Tid-1L protein decreased vascular endothelial growth factor expression and inhibited angiogenesis in vivo and in vitro. These findings propose that Tid-1L may play a critical role in pVHL-mediated tumor suppression by modulating the pVHL-dependent HIF-1α stability.
Individual Variation of Somatic Gene Mutability in Relation to Cancer Susceptibility: Prospective Study on Erythrocyte Glycophorin A Gene Mutations of Atomic Bomb Survivors
Authors: Kyoizumi Seishi , Kusunoki Yoichiro , Hayashi Tomonori , Hakoda Masayuki , Cologne John B. , Nakachi Kei 
Published: September 2005

Abstract
It has previously been reported that hemizygous mutant fraction (Mf) at the glycophorin A (GPA) locus in erythrocytes increased with radiation dose in heterozygotes among Hiroshima and Nagasaki atomic bomb survivors. In the present study, we analyzed the relationship between GPA Mf and cancer risk using newly developed cancers among previously cancer-free subjects whose GPA Mf had been measured between 1988 and 1996. Among 1,723 survivors (1,117 in Hiroshima and 606 in Nagasaki), we identified 186 subjects who developed a first cancer by the end of 2000. We compared the radiation dose responses of GPA Mf between cancer and cancer-free groups using a linear-quadratic model fit by multiple regression analysis in combination with age, sex, and city. The slope of the GPA Mf dose-response curve was significantly higher in the cancer group than in the cancer-free group among Hiroshima subjects. Moreover, no significant difference of GPA Mf between cancer and cancer-free groups was found in unexposed controls in the two cities. The same conclusions were obtained using a linear dose-response model and by further analysis using Cox regression of cancer incidence. These findings suggest that there might be interindividual variation in mutability of somatic genes and that Hiroshima survivors who have higher mutability in response to radiation exposure would be expected to have a higher probability of suffering radiation-related cancer.
Increased Expression of the E3-Ubiquitin Ligase Receptor Subunit βTRCP1 Relates to Constitutive Nuclear Factor-κB Activation and Chemoresistance in Pancreatic Carcinoma Cells
Authors: Müerköster Susanne , Arlt Alexander , Sipos Bence , Witt Maike , Großmann Maike , Klöppel Günter , Kalthoff Holger , Fölsch Ulrich R. , Schäfer Heiner 
Published: September 2005

Abstract
The permanent activation of the transcription factor nuclear factor-κB (NF-κB) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-κB activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit βTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-κB activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with βTRCP1, PT45-P1 cells exhibit an elevated NF-κB activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of βTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-κB activation and chemoresistance. In PancTu-1 cells, βTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1β resulted in an increased expression of βTRCP1, and transfection of this cell line with βTRCP1 induced IL-1β secretion in a NF-κB–dependent fashion. Thus, via its close and mutual link to IL-1β secretion, βTRCP1 expression might substantially contribute to the persistent, IL-1β–dependent activation of NF-κB in pancreatic carcinoma cells. In support of this, βTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-κB. Altogether, our findings of the elevated βTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-κB activation and thereby of chemoresistance.
Elevated Serum Levels of Stromal-Derived Factor-1α Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients
Authors: Zannettino Andrew C.W. , Farrugia Amanda N. , Kortesidis Angela , Manavis Jim , To L. Bik , Martin Sally K. , Diamond Peter , Tamamura Hirokazu , Lapidot Tsvee , Fujii Nobutaka , Gronthos Stan 
Published: September 2005

Abstract
Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1α (SDF-1α) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1α protein and exhibit elevated plasma levels of SDF-1α when compared with normal, age-matched subjects. The level of SDF-1α positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1α in osteoclast precursor recruitment and activation. To examine this further, peripheral blood–derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1α. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1α mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1α was associated with an increase in the expression of a number of osteoclast activation–related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1α by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1α axis may provide an effective means of treatment for MM-induced osteolysis.
Flavonoid Structure-Activity Studies Identify 6-Prenylchrysin and Tectochrysin as Potent and Specific Inhibitors of Breast Cancer Resistance Protein ABCG2
Authors: Ahmed-Belkacem Abdelhakim , Pozza Alexandre , Muñoz-Martínez Francisco , Bates Susan E. , Castanys Santiago , Gamarro Francisco , Di Pietro Attilio , Pérez-Victoria José M. 
Published: September 2005

Abstract
Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 μmol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 μmol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 μmol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.