bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation and limitations to fatty acid oxidation during exercise
Authors: Jeppesen Jacob , Kiens Bente
Published: January 2012

Abstract  Fatty acids (FAs) as fuel for energy utilization during exercise originate from different sources: FAs transported in the circulation either bound to albumin or as triacylglycerol (TG) carried by very low density lipoproteins and FAs from lipolysis of muscle TG stores. Despite a high rate of energy expenditure during high intensity exercise the total FA oxidation is suppressed to below that observed during moderate intensity exercise. Although this has been known for many years, the mechanisms behind this phenomenon are still not fully elucidated. A failure of adipose tissue to deliver sufficient FAs to exercising muscle has been proposed, but evidence is emerging that factors within the muscle might be of more importance. The high rate of glycolysis during high intensity exercise might be the ‘driving force’ via the increased production of acetyl‐CoA, which in turn is trapped by carnitine. This will lead to decreased availability of free carnitine for long chain FA transport into mitochondria. This review summarizes our present view on how FA metabolism is regulated during exercise with a special focus on the limitations in FA oxidation in the transition from moderate to high intensity exercise in humans.
Relationship of Coal-Ash Viscosity to Chemical Composition
Authors: Sage W. L. , McIlroy J. B.
Published: January 2012

This paper discusses information on coal-ash behavior in the temperature range suitable for liquid ash removal and shows how this is related to boiler design and fuel characteristics. A description of a method for predicting slag viscosity from ash analysis and fusion temperatures is given. Also, a method is shown for predicting the flux requirements to obtain desired slag flow characteristics from an unsatisfactory fuel.
Design Control of Overcompression in Rotary-Vane Compressors
Authors: Bransford E. O. , Stein R. A.
Published: January 2012

In its simplest terms, the design of a rotary-vane compressor of a given displacement consists of selecting a particular combination of rotor and cylinder radius, cylinder length, and number of vanes. A study of the rotary-vane compressor has shown that the choice is not entirely arbitrary since thermodynamic and mechanical losses occurring in the compressor are intimately associated with these basic dimensions. One of the more important of these geometrically dependent losses was found to be potentially severe overcompression during the discharge process. In this paper, an equation is developed that describes in generalized form the relation of overcompression to the dimensions of the compressor. This equation is used to evolve an energy-loss criterion for design control of this loss. An example of the application of the criterion to a refrigeration compressor is included.
Throat Tap Nozzles Used for Accurate Flow Measurements
Authors: Cotton K. C. , Westcott J. C.
Published: January 2012

The history of the design, manufacture, calibration, and use of 17 nozzles built by the General Electric Company for accurate flow measurements in conducting performance tests on large steam turbine-generator units are presented.
Optimal Parameters and Characteristics of a Three Degree of Freedom Dynamic Vibration Absorber
Authors: Febbo Mariano
Published: January 2012

The present study is devoted to the determination of the optimal parameters and characteristics of a three degree of freedom dynamic vibration absorber (3 DOF DVA) for the vibration reduction of a plate at a given point. The optimization scheme uses simulated annealing and constrained simulated annealing, which is capable of optimizing systems with a set of constraints. Comparisons between a 3 DOF DVA and multiple (5) 1 DOF DVAs show a better performance of the former for vibration reduction. Regarding the characteristics of the optimal 3 DOF DVA, numerical tests reveal that the absorber is robust under variations of the observation point and for 10% variations of its mass, stiffness and damping. From the analysis of parameter changes of the plate, it is found that the optimal 3 DOF DVA is almost insensitive to a mass change, and sensitive to a change of Young’s modulus for low frequencies. In this case, a decrease in Young’s modulus causes a decrease in its effectiveness, and an increase improves it. The study of the effect of the 3 DOF DVA location on its effectiveness reveals that the requirements of closeness of the absorber to an antinode of the bare primary structure and to the observation point improve its performance. Additionally, for a rotational mode of the 3-DOF DVA about some axis, the effectiveness of the absorber at a given frequency can be notably increased if it is located at a position of the primary system with an in-phase or out-of- phase motion of the attachment points according to the rotational-mode characteristics of the 3-DOF DVA at this frequency.
Thermosolutal Natural Convection in Partially Porous Domains
Authors: Gobin Dominique , Goyeau Benoît
Published: January 2012

In many industrial processes or natural phenomena, coupled heat and mass transfer and fluid flow take place in configurations combining a clear fluid and a porous medium. Since the pioneering work by Beavers and Joseph (1967), the modeling of such systems has been a controversial issue, essentially due to the description of the interface between the fluid and the porous domains. The validity of the so-called one-domain approach—more intuitive and numerically simpler to implement—compared to a two-domain description where the interface is explicitly accounted for, is now clearly assessed. This paper reports recent developments and the current state of the art on this topic, concerning the numerical simulation of such flows as well as the stability studies. The continuity of the conservation equations between a fluid and a porous medium are examined and the conditions for a correct handling of the discontinuity of the macroscopic properties are analyzed. A particular class of problems dealing with thermal and double diffusive natural convection mechanisms in partially porous enclosures is presented, and it is shown that this configuration exhibits specific features in terms of the heat and mass transfer characteristics, depending on the properties of the porous domain. Concerning the stability analysis in a horizontal layer where a fluid layer lies on top of a porous medium, it is shown that the onset of convection is strongly influenced by the presence of the porous medium. The case of double diffusive convection is presented in detail.
Growth of GaSb1−xBix by molecular beam epitaxy
Authors: Song Yuxin , Wang Shumin , Saha Roy Ivy , Shi Peixiong , Hallen Anders
Published: January 2012

Molecular beam epitaxy for GaSb1−xBix is investigated in this article. The growth window for incorporation of Bi in GaSb was found. Strategies of avoiding formation of Bi droplets and enhancing Bi incorporation were studied. The Bi incorporation was confirmed by SIMS and RBS measurements. The Bi concentration in the samples was found to increase with increasing growth temperature and Bi flux. The position of GaSb1−xBix layer peak in XRD rocking curves is found to be correlated to Bi composition. Surface and structural properties of the samples were also investigated. Samples grown on GaSb and GaAs substrates were compared and no apparent difference for Bi incorporation was found.
Tension at the Leading Edge
Authors: Foley John F.
Published: January 2012

The increased membrane tension at the leading edge of a migrating cell maintains cell polarity.
A Genomewide Screen in Schizosaccharomyces pombe for Genes Affecting the Sensitivity of Antifungal Drugs That Target Ergosterol Biosynthesis
Authors: Fang Yue , Hu Lingling , Zhou Xin , Jaiseng Wurentuya , Zhang Ben , Takami Tomonori , Kuno Takayoshi
Published: January 2012

We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a
Schizosaccharomyces pombe
gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-
d
-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs.
S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) Reduce
In Vitro
Inhibition by Fluconazole
Authors: Warrilow Andrew G. S. , Mullins Jonathan G. L. , Hull Claire M. , Parker Josie E. , Lamb David C. , Kelly Diane E. , Kelly Steven L.
Published: January 2012

The effects of S279F and S279Y point mutations in
Candida albicans
CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (

K
s

, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (

K
s

, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC
50
s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher
K
d
values observed.
NagZ-Dependent and NagZ-Independent Mechanisms for β-Lactamase Expression in Stenotrophomonas maltophilia
Authors: Huang Yi-Wei , Hu Rouh-Mei , Lin Cheng-Wen , Chung Tung-Ching , Yang Tsuey-Ching
Published: January 2012

β-
N
-Acetylglucosaminidase (NagZ), encoded by the
nagZ
gene, is a critical enzyme for basal-level
ampC
derepression (
ampC
expression in the absence of β-lactam challenge) in
ampD
and
dacB
mutants of
Pseudomonas aeruginosa
. Three mutants with a phenotype of basal-level L1 and L2 β-lactamase derepression in
Stenotrophomonas maltophilia
have been reported, including KJΔDI (

ampD
I

mutant), KJΔmrcA (
mrcA
mutant), and KJΔDIΔmrcA (

ampD
I

and
mrcA
double mutant). In this study,
nagZ
of
S. maltophilia
was characterized, and its roles in basal-level β-lactamase derepression, induced β-lactamase activities, and β-lactam resistance of KJΔDI, KJΔmrcA, and KJΔDIΔmrcA were evaluated. Expression of the
nagZ
gene was constitutive and not regulated by AmpR, AmpD
I
, AmpN, AmpG, PBP1a, and NagZ. Introduction of Δ
nagZ
into KJΔDI nearly abolished basal-level derepressed β-lactamase activity; conversely, introduction of Δ
nagZ
into KJΔmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for β-lactamase expression in the
S. maltophilia
system, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed β-lactamase activities of KJΔDI and KJΔmrcA, respectively. The contributions of AL1 and AL2 to the induced β-lactamase activities may vary with the types of β-lactams.
nagZ
inactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced β-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced β-lactamase activities. Introduction of Δ
nagZ
into KJ, KJΔDI, KJΔmrcA, and KJΔDIΔmrcA did not significantly change the MICs of the β-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJΔZ and KJΔDIΔZ (
nagZ
mutants).
SQ109 Targets MmpL3, a Membrane Transporter of Trehalose Monomycolate Involved in Mycolic Acid Donation to the Cell Wall Core of Mycobacterium tuberculosis
Authors: Tahlan Kapil , Wilson Regina , Kastrinsky David B. , Arora Kriti , Nair Vinod , Fischer Elizabeth , Barnes S. Whitney , Walker John R. , Alland David , Barry Clifton E. , Boshoff Helena I.
Published: January 2012

SQ109, a 1,2-diamine related to ethambutol, is currently in clinical trials for the treatment of tuberculosis, but its mode of action remains unclear. Here, we demonstrate that SQ109 disrupts cell wall assembly, as evidenced by macromolecular incorporation assays and ultrastructural analyses. SQ109 interferes with the assembly of mycolic acids into the cell wall core of
Mycobacterium tuberculosis
, as bacilli exposed to SQ109 show immediate inhibition of trehalose dimycolate (TDM) production and fail to attach mycolates to the cell wall arabinogalactan. These effects were not due to inhibition of mycolate synthesis, since total mycolate levels were unaffected, but instead resulted in the accumulation of trehalose monomycolate (TMM), the precursor of TDM and cell wall mycolates.
In vitro
assays using purified enzymes showed that this was not due to inhibition of the secreted Ag85 mycolyltransferases. We were unable to achieve spontaneous generation of SQ109-resistant mutants; however, analogs of this compound that resulted in similar shutdown of TDM synthesis with concomitant TMM accumulation were used to spontaneously generate resistant mutants that were also cross-resistant to SQ109. Whole-genome sequencing of these mutants showed that these all had mutations in the essential
mmpL3
gene, which encodes a transmembrane transporter. Our results suggest that MmpL3 is the target of SQ109 and that MmpL3 is a transporter of mycobacterial TMM.
Azole Resistance by Loss of Function of the Sterol Δ
5,6
-Desaturase Gene (
ERG3
) in Candida albicans Does Not Necessarily Decrease Virulence
Authors: Vale-Silva L. A. , Coste A. T. , Ischer F. , Parker J. E. , Kelly S. L. , Pinto E. , Sanglard D.
Published: January 2012

The inactivation of
ERG3
, a gene encoding sterol Δ
5,6
-desaturase (essential for ergosterol biosynthesis), is a known mechanism of
in vitro
resistance to azole antifungal drugs in the human pathogen
Candida albicans. ERG3
inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a
C. albicans
clinical isolate (VSY2) with high-level resistance to azole drugs
in vitro
and an absence of ergosterol but normal filamentation. Sequencing of
ERG3
in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (
erg3-1
) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that
ERG3
inactivation was the mechanism of azole resistance. Additionally, the replacement of both
ERG3
alleles by
erg3-1
in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical
ERG3
mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both
in vitro
and
in vivo
, the
ERG3
-derived mutant of SC5314 was resistant only
in vitro
and was less virulent than the wild type. This suggests that VSY2 compensated for the
in vivo
fitness defect of
ERG3
inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of
ERG3
does not necessarily affect filamentation and virulence.
A Semimechanistic Pharmacokinetic-Enzyme Turnover Model for Rifampin Autoinduction in Adult Tuberculosis Patients
Authors: Smythe Wynand , Khandelwal Akash , Merle Corinne , Rustomjee Roxana , Gninafon Martin , Bocar Lo Mame , Sow Oumou Bah , Olliaro Piero L. , Lienhardt Christian , Horton John , Smith Peter , McIlleron Helen , Simonsson Ulrika S. H.
Published: January 2012

The currently recommended doses of rifampin are believed to be at the lower end of the dose-response curve. Rifampin induces its own metabolism, although the effect of dose on the extent of autoinduction is not known. This study aimed to investigate rifampin autoinduction using a semimechanistic pharmacokinetic-enzyme turnover model. Four different structural basic models were explored to assess whether different scaling methods affected the final covariate selection procedure. Covariates were selected by using a linearized approach. The final model included the allometric scaling of oral clearance and apparent volume of distribution. Although HIV infection was associated with a 30% increase in the apparent volume of distribution, simulations demonstrated that the effect of HIV on rifampin exposure was slight. Model-based simulations showed close-to-maximum induction achieved after 450-mg daily dosing, since negligible increases in oral clearance were observed following the 600-mg/day regimen. Thus, dosing above 600 mg/day is unlikely to result in higher magnitudes of autoinduction. In a typical 55-kg male without HIV infection, the oral clearance, which was 7.76 liters · h
−1
at the first dose, increased 1.82- and 1.85-fold at steady state after daily dosing with 450 and 600 mg, respectively. Corresponding reductions of 41 and 42%, respectively, in the area under the concentration-versus-time curve from 0 to 24 h were estimated. The turnover of the inducible process was estimated to have a half-life of approximately 8 days in a typical patient. Assuming 5 half-lives to steady state, this corresponds to a duration of approximately 40 days to reach the induced state for rifampin autoinduction.
pJIE137 Carrying
bla
CTX-M-62
Is Closely Related to p271A Carrying
bla
NDM-1
Authors: Partridge Sally R. , Paulsen Ian T. , Iredell Jonathan R.
Published: January 2012

Complete sequencing of pJIE137 revealed a backbone closely related to p271A, encoding a novel RepA protein but with a similar organization and up to ∼70% nucleotide identity to IncN plasmids. A region in pJIE137 resembling the IncN CUP regulon is mostly missing from p271A, presumably due to recombination. The class 1 In/Tn and IS
Ecp1-bla
CTX-M-62
transposition unit in pJIE137 and a putative transposon carrying
bla
NDM-1
in p271A are inserted in different locations in the plasmid backbone.
In Vitro
and
In Vivo
Antimalarial Activities of T-2307, a Novel Arylamidine
Authors: Kimura Akiko , Nishikawa Hiroshi , Nomura Nobuhiko , Mitsuyama Junichi , Fukumoto Shinya , Inoue Noboru , Kawazu Shin-ichiro
Published: January 2012

T-2307, a novel arylamidine, has been shown to exhibit broad-spectrum antifungal activities against clinically significant pathogens. Here, we evaluated the
in vitro
and
in vivo
antimalarial activity of T-2307. The 50% inhibitory concentrations (IC
50
s) of T-2307 against
Plasmodium falciparum
FCR-3 and K-1 strains were 0.47 and 0.17 μM, respectively. T-2307 at 2.5 to 10 mg/kg of body weight/day exhibited activity against blood stage and liver stage parasites in rodent malaria models. In conclusion, T-2307 exhibited
in vitro
and
in vivo
antimalarial activity.
The Natural Antimicrobial Peptide Subtilosin Acts Synergistically with Glycerol Monolaurate, Lauric Arginate, and ε-Poly-
l
-Lysine against Bacterial Vaginosis-Associated Pathogens but Not Human Lactobacilli
Authors: Sutyak Noll Katia , Prichard Mark N. , Khaykin Arkady , Sinko Patrick J. , Chikindas Michael L.
Published: January 2012

Subtilosin is a cyclical antimicrobial peptide produced by
Bacillus amyloliquefaciens
that has antimicrobial activity against the bacterial vaginosis-associated human pathogen
Gardnerella vaginalis
. The ability of subtilosin to inhibit
G. vaginalis
alone and in combination with the natural antimicrobial agents glycerol monolaurate (Lauricidin), lauric arginate, and ε-poly-
l
-lysine was tested using a checkerboard approach. Subtilosin was found to act synergistically with all of the chosen antimicrobials. These promising results indicate that lower concentrations of subtilosin in combination with other compounds could effectively be used to inhibit growth of the pathogen, thereby decreasing the risk of developed antimicrobial resistance. This is the first report on the effects of subtilosin combined with other natural antimicrobials against
G. vaginalis
.
Interleukin-17 Drives Pulmonary Eosinophilia following Repeated Exposure to Aspergillus fumigatus Conidia
Authors: Murdock Benjamin J. , Falkowski Nicole R. , Shreiner Andrew B. , Sadighi Akha Amir A. , McDonald Roderick A. , White Eric S. , Toews Galen B. , Huffnagle Gary B.
Published: January 2012

Previous research in our laboratory has demonstrated that repeated intranasal exposure toAspergillus fumigatusconidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper TH2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the TH2 response has begun to resolve. TH17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged withA. fumigatusconidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4+CD25+FoxP3+cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving TH2-type inflammation to repeated inhalation of fungal conidia.
Protective Role ofd-Amino Acid Oxidase against Staphylococcus aureus Infection
Authors: Nakamura Hideaki , Fang Jun , Maeda Hiroshi
Published: January 2012

d-Amino acid oxidase (DAO) is a hydrogen peroxide-generating enzyme that uses ad-amino acid as a substrate. We hypothesized that DAO may protect against bacterial infection, because hydrogen peroxide is one of the most important molecules in the antibacterial defense systems in mammals. We show here that DAO suppressed the growth ofStaphylococcus aureusin a manner that depended on the concentration of DAO andd-amino acidin vitro. Addition of catalase abolished the bacteriostatic activity of DAO. Although DAO plusd-Ala showed less bactericidal activity, addition of myeloperoxidase (MPO) greatly enhanced the bactericidal activity of DAO. Furthermore, DAO was able to utilize bacterial lysate, which containsd-Ala derived from peptidoglycan; this could produce hydrogen peroxide with, in the presence of myeloperoxidase, formation of hypochlorous acid. This concerted reaction of DAO and MPO led to the bactericidal action.In vivoexperiments showed that DAO−/−(mutant) mice were more susceptible toS. aureusinfection than were DAO+/+(wild-type) mice. These results suggest that DAO, together with myeloperoxidase, may play an important role in antibacterial systems in mammals.
Identification of the Cellular Receptor of Clostridium spiroforme Toxin
Authors: Papatheodorou Panagiotis , Wilczek Claudia , Nölke Thilo , Guttenberg Gregor , Hornuss Daniel , Schwan Carsten , Aktories Klaus
Published: January 2012

Clostridium spiroforme
produces the binary actin-ADP-ribosylating toxin CST (
C. spiroforme
toxin), which has been proposed to be responsible for diarrhea, enterocolitis, and eventually death, especially in rabbits. Here we report on the recombinant production of the enzyme component (CSTa) and the binding component (CSTb) of
C. spiroforme
toxin in
Bacillus megaterium
. By using the recombinant toxin components, we show that CST enters target cells via the lipolysis-stimulated lipoprotein receptor (LSR), which has been recently identified as the host cell receptor of the binary toxins
Clostridium difficile
transferase (CDT) and
Clostridium perfringens
iota toxin. Microscopic studies revealed that CST, but not the related
Clostridium botulinum
C2 toxin, colocalized with LSR during toxin uptake and traffic to endosomal compartments. Our findings indicate that CST shares LSR with
C. difficile
CDT and
C. perfringens
iota toxin as a host cell surface receptor.
Overexpression of C-terminally but not N-terminally truncated Myb induces fibrosarcomas: a novel nonhematopoietic target cell for the myb oncogene.
Authors: Press R D , Reddy E P , Ewert D L
Published: January 2012

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb's oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.
The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence.
Authors: Méthot N , Pause A , Hershey J W , Sonenberg N
Published: January 2012

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.
The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.
Authors: Zhan Q , Lord K A , Alamo I , Hollander M C , Carrier F , Ron D , Kohn K W , Hoffman B , Liebermann D A , Fornace A J
Published: January 2012

A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, whic, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggest a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.
Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase.
Authors: Kato J Y , Matsuoka M , Strom D K , Sherr C J
Published: January 2012

The accumulation of assembled holoenzymes composed of regulatory D-type cyclins and their catalytic partner, cyclin-dependent kinase 4 (cdk4), is rate limiting for progression through the G1 phase of the cell cycle in mammalian fibroblasts. Both the synthesis and assembly of D-type cyclins and cdk4 depend upon serum stimulation, but even when both subunits are ectopically overproduced, they do not assemble into complexes in serum-deprived cells. When coexpressed from baculoviral vectors in intact Sf9 insect cells, cdk4 assembles with D-type cyclins to form active protein kinases. In contrast, recombinant D-type cyclin and cdk4 subunits produced in insect cells or in bacteria do not assemble as efficiently into functional holoenzymes when combined in vitro but can be activated in the presence of lysates obtained from proliferating mammalian cells. Assembly of cyclin D-cdk4 complexes in coinfected Sf9 cells facilitates phosphorylation of cdk4 on threonine 172 by a cdk-activating kinase (CAK). Assembly can proceed in the absence of this modification, but cdk4 mutants which cannot be phosphorylated by CAK remain catalytically inactive. Therefore, formation of the cyclin D-cdk4 complex and phosphorylation of the bound catalytic subunit are independently regulated, and in addition to the requirement for CAK activity, serum stimulation is required to promote assembly of the complexes in mammalian cells.
FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis.
Authors: Ellinger-Ziegelbauer H , Hihi A K , Laudet V , Keller H , Wahli W , Dreyer C
Published: January 2012

Orphan receptors of the FTZ-F1-related group of nuclear receptors (xFF1r) were identified in Xenopus laevis by isolation of cDNAs from a neurula stage library. Two cDNAs were found, which encode full length, highly related receptor proteins, xFF1rA and B, whose closet relative known so far is the murine LRH-1 orphan receptor. xFF1rA protein expressed by a recombinant vaccinia virus system specifically binds to FTZ-F1 response elements (FRE; PyCAAGGPyCPu). In cotransfection studies, xFF1rA constitutively activates transcription, in a manner dependent on the number of FREs. The amounts of at least four mRNAs encoding full-length receptors greatly increase between gastrula and early tailbud stages and decrease at later stages. At early tailbud stages, xFTZ-F1-related antigens are found in all nuclei of the embryo.
A mammalian origin of bidirectional DNA replication within the Chinese hamster RPS14 locus.
Authors: Tasheva E S , Roufa D J
Published: January 2012

Two complementary experimental approaches have been used to identify a chromosomal origin of bidirectional DNA replication within or immediately downstream of the Chinese hamster ribosomal protein S14 gene (RPS14). The replication origin, designated oriS14, maps within a 1.6- to 2.0-kbp region of RPS14 that includes the gene's third and fourth introns, exons IV plus V, and approximately 500 bp of proximal downstream flanking DNA. The nucleic acid sequence encoding oriS14 closely resembles the other mammalian chromosomal replication origins whose primary structures are known. It contains DNA binding sites for a large number of transcription factors, replication proteins, and mammalian oncogenes as well as several dinucleotide repeat motifs, an AT-rich region, and a sequence that is likely to bend the DNA. In contrast to the other well-characterized mammalian replication origins, which are autosomal and therefore carried as two copies per somatic cell, oriS14 is encoded by single-copy DNA within a hemizygous segment of chromosome 2q in CHO-K1 cells. Also, other known mammalian replication origins are situated in nontranscribed, intergenic DNA, whereas the DNA sequence encoding oriS14 substantially overlaps the transcribed portion of a constitutively expressed housekeeping gene.
Densely methylated DNA islands in mammalian chromosomal replication origins.
Authors: Tasheva E S , Roufa D J
Published: January 2012

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.
Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene.
Authors: Shibuya H , Kohu K , Yamada K , Barsoumian E L , Perlmutter R M , Taniguchi T
Published: January 2012

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.
Two pathways for serum regulation of the c-fos serum response element require specific sequence elements and a minimal domain of serum response factor.
Authors: Johansen F E , Prywes R
Published: January 2012

The c-fos serum response element (SRE) is necessary and sufficient for induction of the c-fos gene in response to serum and growth factors. This activation is dependent upon serum response factor (SRF), a transcriptional activator which binds the SRE. A factor, p62TCF, which binds in conjunction with SRF to the SRE and which is activated by mitogen-activated protein kinase, has also been implicated in c-fos regulation. By using a reporter gene system with weak SRE mutations that is dependent upon overexpression of SRF for serum induction, we have found that there are at least two pathways for serum induction that converge on the SRE. Loss of TCF binding by mutations in SRF and the SRE did not reduce serum induction of the reporter genes. We have found a pathway for serum induction that is sensitive to mutations in the A/T-containing central sequence of the SRE and which is independent of TCF. When this pathway was mutated, activation was dependent upon TCF binding, demonstrating that TCF can also function in serum induction. Both of the signalling pathways required a minimal domain of SRF. This domain, spanning SRF's DNA binding domain, was sufficient for serum induction when fused to a heterologous transcriptional activation domain.
Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.
Authors: Tanaka M , Herr W
Published: January 2012

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.
Purification and characterization of nucleolin and its identification as a transcription repressor.
Authors: Yang T H , Tsai W H , Lee Y M , Lei H Y , Lai M Y , Chen D S , Yeh N H , Lee S C
Published: January 2012

Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.
The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.
Authors: Sturm N R , Kuras R , Büschlen S , Sakamoto W , Kindle K L , Stern D B , Wollman F A
Published: January 2012

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.
DBF8, an essential gene required for efficient chromosome segregation in Saccharomyces cerevisiae.
Authors: Houman F , Holm C
Published: January 2012

To investigate chromosome segregation in Saccharomyces cerevisiae, we examined a collection of temperature-sensitive mutants that arrest as large-budded cells at restrictive temperatures (L. H. Johnston and A. P. Thomas, Mol. Gen. Genet. 186:439-444, 1982). We characterized dbf8, a mutation that causes cells to arrest with a 2c DNA content and a short spindle. DBF8 maps to chromosome IX near the centromere, and it encodes a 36-kDa protein that is essential for viability at all temperatures. Mutational analysis reveals that three dbf8 alleles are nonsense mutations affecting the carboxy-terminal third of the encoded protein. Since all of these mutations confer temperature sensitivity, it appears that the carboxyl-terminal third of the protein is essential only at a restrictive temperature. In support of this conclusion, an insertion of URA3 at the same position also confers a temperature-sensitive phenotype. Although they show no evidence of DNA damage, dbf8 mutants exhibit increased rates of chromosome loss and nondisjunction even at a permissive temperature. Taken together, our data suggest that Dbf8p plays an essential role in chromosome segregation.
Peripheral neuropathy: pattern recognition for the pragmatist
Authors: Overell James R
Published: January 2012

Long lists of causes of peripheral neuropathy make peripheral nerve disease a dry and uninspiring subject. A simple scheme based on the answers to just six questions should enable the clinician to recognise characteristic patterns, investigate relevant subgroups appropriately, and identify treatable disorders quickly: which systems are involved? What is the distribution of weakness? What is the nature of the sensory involvement? Is there any evidence of upper motor neuron involvement? What is the temporal evolution? Is there any evidence for a hereditary neuropathy? Standard screening investigations suffice for the common length dependent axonal neuropathies while complex presentations need more detailed investigations targeted to their clinical phenotype.
Quasi-classical trajectory calculation of the chemical reaction Ca + CF3Br
Authors: Xia Wenwen , Yao Li , Zhong Haiyang
Published: January 2012

The dynamical properties of the product CaBr for the reaction Ca + CF3Br were studied with the quasi-classical trajectory method based on the extended London–Eyring–Polanyi–Sato potential energy surface. In this paper, the results, such as vibrational distribution, reaction cross section, and rotational alignment of the product CaBr, were calculated and the reaction mechanism has been discussed. The peak value of the vibrational distribution is located at around v = 3 when the collision energy equals 0.55 eV. The result obtained in this paper agrees well with the experimental vibrational result. The reaction cross section and the product rotational alignment all decrease with the increasing collision energy, which changes from 0.1 to 1.0 eV.
Highly efficient one-pot synthesis of trisubstituted imidazoles under catalyst-free conditions
Authors: Azizi Najmadin , Dado Nairreh , Amiri Alireza Khajeh
Published: January 2012

Operationally simple, atom economical, and scalable synthesis of 2,4,5-trisubstituted imidazoles from benzil, aldehydes, and ammonium acetate is shown to proceed readily in methanol with high yield. The scope of the reaction is quite broad; a variety of aromatic and aliphatic activated and unactivated aldehydes have all been shown to be viable substrates for this reaction. Excellent yields and purity were obtained by washing the products with hot ethanol.