bluuebunny/crossref_metadata_2025 huggingface dataset:
LIGHTWEIGHT GEOPOLYMER WOOD COMPOSITE SYNTHESIZED FROM ALKALI-ACTIVATED FLY ASH AND METAKAOLIN
Authors: Sarmin Siti Noorbaini , Welling Johannes
Published: November 2016

This research paper presents the report on the use of wood particle as reinforcement to produce lightweight geopolymer wood composite. In this study, Class F fly ash and metakaolin used as alumino-silicate source were activated by alkaline activator and samples were cured at two different temperatures, 80°C and 20°C. Wood particle, 3 to 5 mm in size was added to fly ash/metakaolin based geopolymers for 10 – 60% solid content, and its influence on the compressive strength has been investigated. The microstructure investigation using field emission scanning electron microscopy (FESEM) illustrated fly ash; metakaolin and wood particle were embedded in a dense alumino-silicate matrix, though there was some unreacted phase occurred. The compressive strength of lightweight geopolymer matrix without wood particle added samples cured at 80°C temperature was 5.4 MPa which significantly increased 45% when the 10% wood particle were added. However, the addition of wood particle from 20 to 60% has not shown a major improvement in compressive strength.  It was found that the density of the lightweight geopolymer composite was decrease relevant to the amount of wood particles addition. It is suggested that wood particle are one of the potential candidates as reinforcement for lightweight geopolymer composite development.
DIFFERENT FAILURE MODES ASSESSMENT TO IMPROVE THE SANDWICH COMPOSITE PANEL STIFFNESS WITH HONEYCOMB CORE FOR MARINE STRUCTURES APPLICATION
Authors: Nakisa Mehdi , Behrouzi Fatemeh , Mobasher Amini Ahmad
Published: November 2016

This research paper focuses on the prediction of different failure modes to improve the sandwich composite panel with honeycomb core for application in marine structures. Marine, automotive and aerospace industries are continually trying to optimize material performance in terms of strength and weight. Success has been achieved through the growth of high performance materials, including fibrous composites such as ceramics, new alloys, and carbon fiber composites and through the use of structural concepts such as sandwich composite panel construction. Sandwich composite panel construction with honeycomb core consists of three components: two facing sheets, the core that fill the space between the facing sheet and the core-to-facing bonding adhesives. The facing sheets of a sandwich panel can be compared to the flanges of an I-beam element, as they carry the bending stresses to which the beam is subjected. With one facing sheet in compression, the other is in tension. Similarly the honeycomb core corresponds to the web of the I-beam that resists the shear loads and vertical compressive load to the face sheet. This paper presents a model for prediction of different failure mode of face sheet and core material. The obtained results of this model were compared with experimental results and presents that it is a simple and good model.
THE EFFECTS OF SPECIAL EVENTS ON REGRESSION FOR SUBCOMPACT CAR SALES IN THAILAND
Authors: Rattanametawee Witchaya , Leenawong Chartchai , Netisopakul Ponrudee
Published: November 2016

This research proposes a method to dealing with multiple linear regression that integrates the seasonality as well as the effects of some special or unanticipated events for sales figures. The method is then applied to the car sales figures in Thailand after having been through the 2011 national big flood and the 2011-2012 government’s initiative tax-incentive program for boosting the automobile industry. Besides Thailand’s Gross Domestic Products (GDP) and the 12-month Loan’s Interest Rate as explanatory variables, seasonal dummy variables along with the proposed special event variables and appropriate event tagging are incorporated. The statistical results obtained from the proposed regression model with seasons and events, compared to the models with neither seasons nor both yields highest adjusted coefficient of determination (R-squre) and accuracy (MAPE).
Additive Manufacturing of Transparent Soda-Lime Glass Using a Filament-Fed Process
Authors: Luo Junjie , Gilbert Luke J. , Qu Chuang , Landers Robert G. , Bristow Douglas A. , Kinzel Edward C.
Published: November 2016

There are many scientific and engineering applications of transparent glass including optics, communications, electronics, and hermetic seals. However, there has been minimal research toward the additive manufacturing (AM) of transparent glass parts. This paper describes and demonstrates a filament-fed technique for AM of transparent glass. A transparent glass filament is melted by a CO2 laser and solidifies as the workpiece is translated relative to the stationary laser beam. To prevent thermal shock, the workpiece rests on a heated build platform. In order to obtain optically transparent parts, several challenges must be overcome, notably producing index homogeneity and avoiding bubble formation. The effects of key process parameters on the morphology and transparency of the printed glass are explored experimentally. These results are compared to a low-order model relating the process parameters to the temperature of the molten region, which is critical to the quality of the deposited glass. At lower temperatures, the glass is not fully melted, resulting in index variations in the final part, while at higher temperatures, phase separation introduces bubbles and other defects into the part. The correct process avoids these issues and deposits optically transparent glass.
Synthesis of Point Planar Elastic Behaviors Using Three-Joint Serial Mechanisms of Specified Construction
Authors: Huang Shuguang , Schimmels Joseph M.
Published: November 2016

This paper presents methods for the realization of 2 × 2 translational compliance matrices using serial mechanisms having three joints, each either revolute or prismatic and each with selectable compliance. The geometry of the mechanism and the location of the compliance frame relative to the mechanism base are each arbitrary but specified. Necessary and sufficient conditions for the realization of a given compliance with a given mechanism are obtained. We show that, for an appropriately constructed serial mechanism having at least one revolute joint, any single 2 × 2 compliance matrix can be realized by properly choosing the joint compliances and the mechanism configuration. For each type of three-joint combination, requirements on the redundant mechanism geometry are identified for the realization of every point planar elastic behavior at a given location, just by changing the mechanism configuration and the joint compliances.
Numerical Determination of Pseudobreathers of a Three-Dimensional Spherically Symmetric Wave Equation
Authors: Karkowski Janusz
Published: November 2016

A numerical method for finding spherically symmetric pseudobreathers of a nonlinear wave equation is presented. The algorithm, based on pseudospectral methods, is applied to find quasi-periodic solutions with force terms being continuous approximations of the signum function. The obtained pseudobreathers slowly radiate energy and decay after some (usually long) time depending on the period that characterizes (unambiguously) the initial configuration.
Traveling Wave Solutions of a Two-Component Dullin–Gottwald–Holm System
Authors: Zhong Jiyu , Deng Shengfu
Published: November 2016

In this paper, we investigate the traveling wave solutions of a two-component Dullin–Gottwald–Holm (DGH) system. By qualitative analysis methods of planar systems, we investigate completely the topological behavior of the solutions of the traveling wave system, which is derived from the two-component Dullin–Gottwald–Holm system, and show the corresponding phase portraits. We prove the topological types of degenerate equilibria by the technique of desingularization. According to the dynamical behaviors of the solutions, we give all the bounded exact traveling wave solutions of the system, including solitary wave solutions, periodic wave solutions, cusp solitary wave solutions, periodic cusp wave solutions, compactonlike wave solutions, and kinklike and antikinklike wave solutions. Furthermore, to verify the correctness of our results, we simulate these bounded wave solutions using the software maple version 18.
Synthetic Organotellurium Compounds Sensitize Drug-Resistant Candida albicans Clinical Isolates to Fluconazole
Authors: Reis de Sá L. F. , Toledo F. T. , Gonçalves A. C. , Sousa B. A. , dos Santos A. A. , Brasil P. F. , Duarte da Silva V. A. , Tessis A. C. , Ramos J. A. , Carvalho M. A. , Lamping E. , Ferreira-Pereira A.
Published: November 2016

Invasive
Candida albicans
infections are a serious health threat for immunocompromised individuals. Fluconazole is most commonly used to treat these infections, but resistance due to the overexpression of multidrug efflux pumps is of grave concern. This study evaluated the ability of five synthetic organotellurium compounds to reverse the fluconazole resistance of
C. albicans
clinical isolates. Compounds 1 to 4, at <10 μg/ml, ameliorated the fluconazole resistance of
Saccharomyces cerevisiae
strains overexpressing the major
C. albicans
multidrug efflux pumps Cdr1p and Mdr1p, whereas compound 5 only sensitized Mdr1p-overexpressing strains to fluconazole. Compounds 1 to 4 also inhibited efflux of the fluorescent substrate rhodamine 6G and the ATPase activity of Cdr1p, whereas all five of compounds 1 to 5 inhibited Nile red efflux by Mdr1p. Interestingly, all five compounds demonstrated synergy with fluconazole against efflux pump-overexpressing fluconazole-resistant
C. albicans
clinical isolates, isolate 95-142 overexpressing
CDR1
and
CDR2
, isolate 96-25 overexpressing
MDR1
and
ERG11
, and isolate 12-99 overexpressing
CDR1
,
CDR2
,
MDR1
, and
ERG11
. Overall, organotellurium compounds 1 and 2 were the most promising fluconazole chemosensitizers of fluconazole-resistant
C. albicans
isolates. Our data suggest that these novel organotellurium compounds inhibit pump efflux by two very important and distinct families of fungal multidrug efflux pumps: the ATP-binding cassette transporter Cdr1p and the major facilitator superfamily transporter Mdr1p.
Pharmacodynamics of Voriconazole against Wild-Type and Azole-Resistant Aspergillus flavus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis
Authors: Rudramurthy Shivaprakash M. , Seyedmousavi Seyedmojtaba , Dhaliwal Manpreet , Chakrabarti Arunaloke , Meis Jacques F. , Mouton Johan W.
Published: November 2016

Invasive aspergillosis (IA) due to
Aspergillus flavus
is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in
Aspergillus
spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated
A. flavus
infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the
cyp51C
gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An
F
test showed the 50% effective doses to be significantly different from each other (
P
= 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI
50
) required being negatively and log-linearly related to the MIC (
P
= 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice.
β-Lactamase Inhibitors Enhance the Synergy between β-Lactam Antibiotics and Daptomycin against Methicillin-Resistant Staphylococcus aureus
Authors: Henson Karl Evans R. , Yim Juwon , Smith Jordan R. , Sakoulas George , Rybak Michael J.
Published: November 2016

The evidence for using combination therapy for the treatment of serious methicillin-resistant
Staphylococcus aureus
(MRSA) infections is growing. In this study, we investigated the synergistic effect of daptomycin (DAP) combined with piperacillin-tazobactam and ampicillin-sulbactam against MRSA in time-kill experiments. Six of eight strains demonstrated synergy between DAP and the β-lactam–β-lactamase inhibitor (BLI) combination. In 5/8 strains, the synergy occurred only in the presence of the BLI, highlighting a role for BLIs in peptide–β-lactam synergy.
Substantial Targeting Advantage Achieved by Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model
Authors: Landersdorfer Cornelia B. , Nguyen Tri-Hung , Lieu Linh Thuy , Nguyen Gary , Bischof Robert J. , Meeusen Els N. , Li Jian , Nation Roger L. , McIntosh Michelle P.
Published: November 2016

Colistin, administered as its inactive prodrug colistin methanesulfonate (CMS), is often used in multidrug-resistant Gram-negative pulmonary infections. The CMS and colistin pharmacokinetics in plasma and epithelial lining fluid (ELF) following intravenous and pulmonary dosing have not been evaluated in a large-animal model with pulmonary architecture similar to that of humans. Six merino sheep (34 to 43 kg body weight) received an intravenous or pulmonary dose of 4 to 8 mg/kg CMS (sodium) or 2 to 3 mg/kg colistin (sulfate) in a 4-way crossover study. Pulmonary dosing was achieved via jet nebulization through an endotracheal tube cuff. CMS and colistin were quantified in plasma and bronchoalveolar lavage fluid (BALF) samples by high-performance liquid chromatography (HPLC). ELF concentrations were calculated via the urea method. CMS and colistin were comodeled in S-ADAPT. Following intravenous CMS or colistin administration, no concentrations were quantifiable in BALF samples. Elimination clearance was 1.97 liters/h (4% interindividual variability) for CMS (other than conversion to colistin) and 1.08 liters/h (25%) for colistin. On average, 18% of a CMS dose was converted to colistin. Following pulmonary delivery, colistin was not quantifiable in plasma and CMS was detected in only one sheep. Average ELF concentrations (standard deviations [SD]) of formed colistin were 400 (243), 384 (187), and 184 (190) mg/liter at 1, 4, and 24 h after pulmonary CMS administration. The population pharmacokinetic model described well CMS and colistin in plasma and ELF following intravenous and pulmonary administration. Pulmonary dosing provided high ELF and low plasma colistin concentrations, representing a substantial targeting advantage over intravenous administration. Predictions from the pharmacokinetic model indicate that sheep are an advantageous model for translational research.
Phase I Clinical Trial Results of Auranofin, a Novel Antiparasitic Agent
Authors: Capparelli Edmund V. , Bricker-Ford Robin , Rogers M. John , McKerrow James H. , Reed Sharon L.
Published: November 2016

Under an NIH priority to identify new drugs to treat class B parasitic agents, we performed high-throughput screens, which identified the activity of auranofin (Ridaura) against
Entamoeba histolytica
and
Giardia intestinalis
, major causes of water- and foodborne outbreaks. Auranofin, an orally administered, gold (Au)-containing compound that was approved by the FDA in 1985 for treatment of rheumatoid arthritis, was effective
in vitro
and
in vivo
against
E. histolytica
and both metronidazole-sensitive and -resistant strains of
Giardia
. We now report the results of an NIH-sponsored phase I trial to characterize the pharmacokinetics (PK) and safety of auranofin in healthy volunteers using modern techniques to measure gold levels. Subjects received orally 6 mg (p.o.) of auranofin daily, the recommended dose for rheumatoid arthritis, for 7 days and were followed for 126 days. Treatment-associated adverse events were reported by 47% of the subjects, but all were mild and resolved without treatment. The mean gold maximum concentration in plasma (
C
max
) at day 7 was 0.312 μg/ml and the half-life (
t
1/2
) 35 days, so steady-state blood levels would not be reached in short-term therapy. The highest concentration of gold, 13 μM (auranofin equivalent), or more than 25× the 50% inhibitory concentration (IC
50
) for
E. histolytica
and 4× that for
Giardia
, was in feces at 7 days. Modeling of higher doses (9 and 21 mg/day) was performed for systemic parasitic infections, and plasma gold levels of 0.4 to 1.0 μg/ml were reached after 14 days of treatment at 21 mg/day. This phase I trial supports the idea of the safety of auranofin and provides important PK data to support its potential use as a broad-spectrum antiparasitic drug. (This study has been registered at ClinicalTrials.gov under identifier NCT02089048.)
Sustained Nitric Oxide-Releasing Nanoparticles Interfere with Methicillin-Resistant Staphylococcus aureus Adhesion and Biofilm Formation in a Rat Central Venous Catheter Model
Authors: Mihu Mircea Radu , Cabral Vitor , Pattabhi Rodney , Tar Moses T. , Davies Kelvin P. , Friedman Adam J. , Martinez Luis R. , Nosanchuk Joshua D.
Published: November 2016

Staphylococcus aureus
is frequently isolated in the setting of infections of indwelling medical devices, which are mediated by the microbe's ability to form biofilms on a variety of surfaces. Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in patients with prolonged hospitalizations. In this study, we demonstrate that sustained nitric oxide-releasing nanoparticles (NO-np) interfere with
S. aureus
adhesion and prevent biofilm formation on a rat central venous catheter (CVC) model of infection. Confocal and scanning electron microscopy showed that NO-np-treated staphylococcal biofilms displayed considerably reduced thicknesses and bacterial numbers compared to those of control biofilms
in vitro
and
in vivo
, respectively. Although both phenotypes, planktonic and biofilm-associated staphylococci, of multiple clinical strains were susceptible to NO-np, bacteria within biofilms were more resistant to killing than their planktonic counterparts. Furthermore, chitosan, a biopolymer found in the exoskeleton of crustaceans and structurally integrated into the nanoparticles, seems to add considerable antimicrobial activity to the technology. Our findings suggest promising development and translational potential of NO-np for use as a prophylactic or therapeutic against bacterial biofilms on CVCs and other medical devices.
Model of Host-Pathogen Interaction Dynamics Links
In Vivo
Optical Imaging and Immune Responses
Authors: Ale Angelique , Crepin Valerie F. , Collins James W. , Constantinou Nicholas , Habibzay Maryam , Babtie Ann C. , Frankel Gad , Stumpf Michael P. H.
Published: November 2016

Tracking disease progression
in vivo
is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for
in vivo
tracking of bacterial population development and therapeutic response. For a precise understanding of
in vivo
imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of
Citrobacter rodentium
infection, a mouse model for enteropathogenic
Escherichia coli
(EPEC). We connect
in vivo
disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.
Microbial Translocation Associated with an Acute-Phase Response and Elevations in MMP-1, HO-1, and Proinflammatory Cytokines in Strongyloides stercoralis Infection
Authors: Rajamanickam Anuradha , Munisankar Saravanan , Bhootra Yukthi , Dolla Chandrakumar , Nutman Thomas B. , Babu Subash
Published: November 2016

Microbial translocation, characterized by elevated levels of lipopolysaccharide (LPS) and related markers, is a common occurrence in HIV and some parasitic infections. This is usually associated with extensive inflammation and immune activation. To examine the occurrence of microbial translocation and the associated inflammatory response in asymptomatic
Strongyloides stercoralis
infection, we measured the plasma levels of LPS and other microbial translocation markers, acute-phase proteins, inflammatory markers, and proinflammatory cytokines in individuals with (infected [INF]) or without (uninfected [UN])
S. stercoralis
infections. Finally, we also measured the levels of all of these markers in INF individuals following treatment of
S. stercoralis
infection. We show that INF individuals exhibit significantly higher plasma levels of microbial translocation markers (LPS, soluble CD14 [sCD14], intestinal fatty acid-binding protein [iFABP], and endotoxin core IgG antibody [EndoCAb]), acute-phase proteins (α-2 macroglobulin [α-2M], C-reactive protein [CRP], haptoglobin, and serum amyloid protein A [SAA]), inflammatory markers (matrix metalloproteinase 1 [MMP-1] and heme oxygenase 1 [HO-1]), and proinflammatory cytokines (interleukin-6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1], and IL-1β) than do UN individuals. INF individuals exhibit significantly decreased levels of tissue inhibitor of metalloproteinases 4 (TIMP-4). Following treatment of
S. stercoralis
infection, the elevated levels of microbial translocation markers, acute-phase proteins, and inflammatory markers were all diminished. Our data thus show that
S. stercoralis
infection is characterized by microbial translocation and accompanying increases in levels of acute-phase proteins and markers of inflammation and provide data to suggest that microbial translocation is a feature of asymptomatic
S. stercoralis
infection and is associated with an inflammatory response.
Rhizobium meliloti suhR suppresses the phenotype of an Escherichia coli RNA polymerase sigma 32 mutant
Authors: Bent A F , Signer E R
Published: November 2016

sigma 32, the product of the Escherichia coli rpoH locus, is an alternative RNA polymerase sigma factor utilized to express heat shock genes upon a sudden rise in temperature. E. coli K165 [rpoH165(Am) supC(Ts)] is temperature sensitive for growth and does not induce heat shock protein synthesis. We have isolated a locus from Rhizobium meliloti called suhR that allows E. coli K165 to grow at high temperature and induce heat shock protein synthesis. R. meliloti suhR mutants were viable and symbiotically effective. suhR was found to have no DNA or derived amino acid sequence similarity to the genes of previously sequenced sigma factors or other data base entries, although a helix-turn-helix DNA-binding protein motif is present. suhR did not restore the phenotypic defects of delta rpoH E. coli; suppression of the E. coli K165 phenotype is thus likely to involve E. coli sigma 32. Western immunoblots showed that suhR caused an approximately twofold elevation of sigma 32 levels in K165; RNA blots indicated that rpoH mRNA level and stability were not altered. Stabilization of sigma 32 protein and increased rpoH mRNA translation are thus the most probable mechanisms of suppression.
Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria
Authors: Moreno E , Stackebrandt E , Dorsch M , Wolters J , Busch M , Mayer H
Published: November 2016

On the basis of ribosomal 16S sequence comparison, Brucella abortus has been found to be a member of the alpha-2 subdivision of the class Proteobacteria (formerly named purple photosynthetic bacteria and their nonphototrophic relatives). Within the alpha-2 subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the faculty or the obligation of living in close association to eucaryotic cells. The composition of Brucella lipid A suggests a close phylogenetical relationship with members of the alpha-2 group. The chemical analysis of the lipid A fraction revealed that Brucella species contain both glucosamine and diaminoglucose, thus suggesting the presence of a so-called mixed lipid A type. The serological analysis with polyclonal and monoclonal antibodies is in agreement with the existence of mixed lipid A type in B. abortus. The amide-linked fatty acid present as acyl-oxyacyl residues were 3-O-C(16:0)12:0, 3-O-C(16:0)13:0, 3-O-C(16:0)14:0, and 3-O-C(18:0)14:0. The only amide-linked unsubstituted fatty acid detected was 3-OH-C16:0. The ester-linked fatty acids are 3-OH-C16:0, 3-OH-C18:0, C16:0, C17:0, and C18:0. Significant amounts of the large-chain 27-OH-C28:0 were detected together with traces of 25-OH-C26:0 and 29-OH-C30:0. Comparison of the Brucella lipid composition with that of the other Proteobacteria also suggests a close phylogenetical relationship with members of the alpha-2 subdivision. The genealogical grouping of Brucella species with pericellular and intracellular plant and animal pathogens as well as with intracellular plant symbionts suggests a possible evolution of Brucella species from plant-arthropod-associated bacteria.
Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons
Authors: Vanooteghem J C , Cornelis G R
Published: November 2016

We sequenced the minimum replication region of the virulence plasmid pYVe439-80 from a serogroup O:9 Yersinia enterocolitica. This sequence is 68% homologous on a 1,873-nucleotide stretch to the sequence of the RepFIIA replicon of the resistance plasmid R100. The sequence contains two open reading frames, repA and repB, encoding proteins of 33,478 and 9,568 daltons, respectively. The amino acid sequences of the two proteins are 77 and 55% identical, respectively, to proteins RepA1 and RepA2 of the R100 replicon. Analysis of minicells transformed with a copy number mutant demonstrated that the replication region of pYVe439-80 directs the synthesis of a 33-kilodalton protein. Disruption of repA, encoding this protein, abolished replication. Two regions of pYVe439-80 are 76 and 70% homologous, respectively, to the copy number control antisense RNA and to the origin of replication region of R100. A mutation introduced in the pYVe439-80 DNA corresponding to the R100 sequence encoding the copy number control antisense RNA resulted in an increase in copy number, indicating a functional homology between the two replicons.
Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C
Authors: Costantino H R , Brown S H , Kelly R M
Published: November 2016

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.
Translocation and compartmentalization of Escherichia coli hemolysin (HlyA)
Authors: Oropeza-Wekerle R L , Speth W , Imhof B , Gentschev I , Goebel W
Published: November 2016

Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.
Unusual evolution of a superoxide dismutase-like gene from the extremely halophilic archaebacterium Halobacterium cutirubrum
Authors: May B P , Dennis P P
Published: November 2016

The archaebacterium Halobacterium cutirubrum contains a single detectable, Mn-containing superoxide dismutase, which is encoded by the sod gene (B. P. May and P. P. Dennis, J. Biol. Chem. 264:12253-12258, 1989). The genome of H. cutirubrum also contains a closely related sod-like gene (slg) of unknown function that has a pattern of expression different from that of sod. The four amino acid residues that bind the Mn atom are conserved, but the flanking regions of the two genes are unrelated. Although the genes have 87% nucleotide sequence identity, the proteins they encode have only 83% amino acid sequence identity. Mutations occur randomly at the first, second, and third codon positions, and transversions outnumber transitions. Most of the mutational differences between the two genes are confined to two limited regions; other regions totally lack differences. These two gene sequences are apparently in the initial stage of divergent evolution. Presumably, this divergence is being driven by strong selection at the molecular level for either acquisition of new functions or partition and refinement of ancestral functions in one or both of the respective gene products.
Cloning and in vivo and in vitro regulation of cyclic AMP-dependent carbon starvation genes from Escherichia coli
Authors: Blum P H , Jovanovich S B , McCann M P , Schultz J E , Lesley S A , Burgess R R , Matin A
Published: November 2016

The regulation of three Escherichia coli carbon starvation (cst) genes fused to lacZ was examined. Expression of these genes is induced by starvation for a carbon source. The role of carbon and cyclic AMP (cAMP) availability and of an altered-function crp mutation were investigated for their effect on cst expression in vivo. The experiments indicated that cAMP concentrations controlled the absolute expression of one cst fusion, but the other two cst fusions were dependent upon some component not present in exponentially growing cells under conditions of glucose excess, even when cAMP was added. To examine the regulation of these genes in further detail, the three cst::lacZ fusions were cloned on multicopy plasmids. All three cst::lacZ fusions retained their inducible regulatory phenotype in the multicopy state. Analysis of the expression of the cloned cst::lacZ fusions in an in vitro-coupled transcription-translation cell-free system demonstrated that the predominant promoter(s) present on each cloned DNA was dependent on sigma 70 for expression. In vitro cAMP titration curves indicated that this molecule was necessary and sufficient for the expression of one fusion but not sufficient for the second fusion, while the third fusion exhibited constitutive levels of expression in vitro. The results are discussed in the context of the E. coli carbon starvation response.
Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis
Authors: Bedouelle H , Guez V , Vidal-Cros A , Hermann M
Published: November 2016

The tyrS genes from Escherichia coli and Bacillus stearothermophilus were toxic to E. coli when they were carried by plasmids with very high copy numbers (pEMBL8 and pEMBL9). We quantified this effect by comparing the efficiencies of plating of E. coli derivatives harboring recombinant plasmids in various experimental conditions. The toxicity was apparent at both 30 and 37 degrees C. It increased with the growth temperature, the strength of the tyrS promoter, and the copy number of the plasmidic vector. Two- to threefold enhancement of tyrS expression raised the toxicity 300-fold. Point mutations in tyrS that prevent interaction between its product, tyrosyl-tRNA synthetase, and tRNA(Tyr) but do not alter the rate of formation of tyrosyl-adenylate abolished the toxicity. Thus, the toxic effect was due to high cellular levels of synthetase activity. At 30 degrees C, the cellular concentration of tyrosyl-tRNA synthetase reached 55% of that of soluble proteins and led to decreased beta-galactosidase stability. We discuss possible causes of this toxic effect and describe its applications to the study of the recognition and interaction between the synthetase and tRNA(Tyr).
The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators
Authors: Debarbouille M , Arnaud M , Fouet A , Klier A , Rapoport G
Published: November 2016

The expression of the Bacillus subtilis sacPA operon is induced by sucrose. A DNA fragment containing the upstream region of this operon was cloned. This fragment contains a promoter from which the operon is expressed. This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B. subtilis sacB gene. Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene. A similar motif is also present in the bgl operon of Escherichia coli. The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon. DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced. The sacT gene product is very similar to the B. subtilis sacY and to the E. coli bglG gene products. The constitutive sacT30 mutation was identified. It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY. These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.
Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I
Authors: Goldschmidt R M , Thoren-Gordon M , Curtiss R
Published: November 2016

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.
Growth medium-independent genetic competence mutants of Bacillus subtilis
Authors: Dubnau D , Roggiani M
Published: November 2016

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.
Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E
Authors: Witte A , Wanner G , Bläsi U , Halfmann G , Szostak M , Lubitz W
Published: November 2016

Biochemical and genetic studies have suggested that a transmembrane tunnel structure penetrating the inner and outer membranes is formed during the lytic action of bacteriophage phi X174 protein E. In this study we directly visualized the lysis tunnel by using high-magnification scanning and transmission electron microscopy.
Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae
Authors: Kinney A J , Carman G M
Published: November 2016

CDP-diacylglycerol synthase, phosphatidylinositol synthase, and phosphatidylinositol kinase activities were associated with post-Golgi apparatus secretory vesicles destined for the plasma membrane of Saccharomyces cerevisiae. These results suggest that the plasma membrane is capable of synthesizing both CDP-diacylglycerol and phosphatidylinositol as well as phosphorylating phosphatidylinositol.
Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus
Authors: Han T J , Chai T J
Published: November 2016

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.
Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790
Authors: Kariyama R , Shockman G D
Published: November 2016

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.
Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae
Authors: Larson T G , Goodgal S H
Published: November 2016

Evidence is presented indicating that a donor DNA processing step of the Haemophilus influenzae transformation pathway is blocked in the Com-101 mutant. Additional data are presented suggesting that, as in the Rec-2 strain, the donor DNA remains associated with the H. influenzae envelope.
Dependence of oxygen-tolerant nitrogenase activity on divalent cations in Azotobacter vinelandii
Authors: Peterson J B
Published: November 2016

Nitrogenase activity of washed Azotobacter vinelandii cells was enhanced by the addition of Ca2+ and Mg2+, and the enhancement increased with the O2 concentration. In assays provided with a level of O2 that was initially supraoptimal and inhibitory to nitrogenase activity, the addition of Ca2+ or Mg2+ affected both the maximum respiration rate (Vmax) of the cells and the apparent affinity [KS(O2)] of cell respiration for O2. Changes in these parameters correlated with changes in nitrogenase activity. Aeration-dependent increases in Vmax and KS(O2) were inhibited by rifampin and chloramphenicol and were also observed in ammonium-grown cultures.
Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide
Authors: Bowden G A , Baneyx F , Georgiou G
Published: November 2016

beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.
Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis
Authors: Eisen J A , Smith S W , Cavanaugh C M
Published: November 2016

The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.
In vivo studies of the role of SecA during protein export in Escherichia coli
Authors: Chun S Y , Randall L L
Published: November 2016

SecA is found in Escherichia coli both tightly associated with the cytoplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R. J. Cabelli, K. M. Dolan, L. Qian, and D. B. Oliver, J. Biol. Chem. 266:24420-24427, 1991; D. B. Oliver and J. Beckwith, Cell 30:311-319, 1982; W. Wickner, A. J. M. Driessen, and F.-U. Hartl, Annu. Rev. Biochem. 60:101-124, 1991). Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of maltose-binding protein, an exported, periplasmic protein. In addition, under conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the distribution of precursor maltose-binding protein changes in parallel. These results support the idea that cytosolic SecA plays a role in export. With the aim of determining the roles of the multiple binding sites for ATP on SecA, we compared the export defect in a culture of E. coli expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide. The results indicate that the mutational change and treatment with sodium azide inhibit export by affecting different steps in the cycle of ATP binding and hydrolysis by SecA.