bluuebunny/crossref_metadata_2025 huggingface dataset:
Microfilaments and cellular signal transduction: Effect of cytochalasin D on the production of cAMP, inositol phosphates, and on calcium movements in rat parotid glands
Authors: Huleux Claire , Dreux Catherine , Imhoff Viviane , Chambaut‐Guerin Anne‐Marie , Rossignol Bernard
Published: February 2012

Summary— In rat parotid glands, the involvement of the microfilament system in the cellular signal transmission mechanism was tested by measuring the effect of cytochalasin D (which disturbs the microfilament system) on the production of intracellular second messengers. Cytochalasin D (CD) did not affect unstimulated calcium movements (measured by the 45Ca efflux technique) or inositol phosphate production or cAMP accumulation. Neither did it modify the generation of intracellular second messengers induced by activation of the cholinergic muscarinic receptor (calcium and inositol phosphates). CD dit not affect the cAMP accumulation induced by the activation of the β‐adrenergic receptor whereas it strongly inhibited the calcium movements induced by activation of the same receptor. These data suggest that, in rat parotid glands, calcium movements, induced by β‐adrenergic receptor stimulation need an intact microfilament system to occur, whereas the muscarinic pathway (via IP3) does not.
Liquid crystalline phases of sonicated type I collagen
Authors: Giraud‐Guille Marie‐Madeleine
Published: February 2012

Summary— The assembly properties of concentrated solutions of type I collagen molecules are compared before and after a 5‐min sonication, breaking the 300‐nm triple helices into short segments of about 20 nm, with a strong polydispersity. The collagen concentration of these solutions, sonicated or not, was increase up to 100 mg/ml by slow evaporation of the solvent. Whereas the non‐sonicated solutions remain isotropic, the sonicated solutions transform after a few hours into a twisted liquid crystalline phase, well recognizable in polarizing microscopy. The evidence of a twisted assembly of collagen triple helices in vitro is new and relevant in a biological context since it was reported in various collagen matrices.
Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL‐60 and U 937 cells
Authors: Capeillère‐Blandin Chantal , Chauvet Genevieve , Tresset Françoise , Descamps‐Latscha Beatrice
Published: February 2012

Summary— The development of cytochrome b558 Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL‐60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFNγ. O2− release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin‐amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol‐amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2− production is closely related to Cyt b but not to MPO specific contents. In differentiated HL‐60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFNγ to the RA treatment. However, marked differences in the O2− production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA‐stimulated O2− production is rather low ie 100 and 20 times less in granulocytic HL‐60 and monocyto‐macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte‐macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2− production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFNγ and/or with RA.
Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA
Authors: Fournier Franck , Jdaïâa Hassan , Lory Philippe , Brûlé Gérard , Curgy Jean‐Jacques , Guilbault Pierre
Published: February 2012

Summary— Using the whole cell voltage‐clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of IBa could be detected (exogenous IBa). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous IBa could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous IBa was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.
Calbindin D28k is essentially located in the colonic part of the toad intestine
Authors: Parmentier Marc
Published: February 2012

Summary— The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium‐binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine‐like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca2+ active transport.
The plasma membrane of Entamoeba histolytica: structure and dynamics*
Authors: Espinosa‐Cantellano Martha , Martínez‐Palomo Adolfo
Published: February 2012

Summary— The plasma membrane components of the parasitic protozoan Entamoeba histolytica, the causative agent of human invasive amebiasis, have been biochemically and immunologically characterized during the last decade. In addition, genes coding for certain surface proteins have been cloned. In spite of these advances, a unified characterization of plasma membrane antigenic components of the parasite is still required for badly needed advancements in the design of useful diagnostic, epidemiologic, and immunoprophylactic tools. Here we review current knowledge on this issue and address the problem of the considerable variation in the electrophoretic profiles of plasma membrane proteins obtained by different groups. In addition, the differences in the degree of recognition of reported membrane antigens with human immune sera, and the diverse interpretations concerning the possible functions of the surface molecules characterized are discussed. A comparative analysis of plasma membrane proteins of E histolytica trophozoites using three different isolation methods revealed that it is possible to select for specific membrane proteins, depending on the lysis conditions. In our view, the method of Calderón and Avila [13] preserves more proteins than other methods tested [3, 22]. Using sera from recent cases of invasive amebiasis studied by several laboratories in various geographical areas, a basic antigenic pattern of 11 principal proteins with molecular weights of 220, 170, 150, 125, 97, 80, 60, 45, 20 and 9 kDA was established for the pathogenic E histolytica strain HM1:IMSS, used by most research groups.
DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved
Authors: Counis Marie‐France , Chaudun Elisabeth , Courtois Yves , Allinquant Bernadette
Published: February 2012

Summary— Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix [18, 40]. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5–15 mM) to hydrolyze the DNA substrate while the 40 kDa‐activity is inhibited by added divalent cations (5–15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.
Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells
Authors: Bigazzi Massimo
Published: February 2012

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro‐graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus‐associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR‐proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P‐proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.
Clinical and Immunomodulatory Effects of Toceranib Combined with Low‐Dose Cyclophosphamide in Dogs with Cancer
Authors: Mitchell L. , Thamm D.H. , Biller B.J.
Published: February 2012

Background

Tyrosine kinase inhibitors (TKIs) and metronomic dosing of cyclophosphamide (CYC) can improve tumor control by suppression of regulatory T cells (Treg) and restoration of T cell‐mediated immune responses in mice and humans. The immunomodulatory effects of the TKI toceranib, as a single agent or in combination with metronomic CYC, have not been previously investigated in dogs.

Hypothesis

The primary objectives of this study were to determine the effects of toceranib and metronomic CYC treatment on lymphocyte subsets including Treg and on interferon‐gamma (IFN‐γ) secretion in dogs with cancer. We hypothesized that toceranib would selectively decrease Treg numbers and increase IFN‐γ production and that addition of CYC would further enhance these effects.

Animals

Fifteen client‐owned dogs with advanced tumors were entered into a prospective clinical trial.

Methods

Dogs received toceranib at 2.75 mg/kg once every other day. After 2 weeks, oral CYC was added at 15 mg/m2 daily. Numbers of Treg and lymphocyte subsets were measured in blood by flow cytometry during the 8‐week study period. Serum concentrations of IFN‐γ were measured by ELISA.

Results

Administration of toceranib significantly decreased the number and percentage of Treg in the peripheral blood of dogs with cancer. Dogs receiving toceranib and CYC demonstrated a significant increase in serum concentrations of IFN‐γ, which was inversely correlated with Treg numbers after 6 weeks of combination treatment.

Conclusions

In addition to antitumor effects, these data support further investigations into the immunomodulatory effects of toceranib, administered alone or in combination with CYC in dogs with cancer.
Effect of chronic cervical ganglionectomy on the spontaneous variability of internal carotid blood flow in the conscious rat
Authors: Revel Aurélia , Gallet Clément , Oréa Valérie , Chapuis Bruno , Barrès Christian , Julien Claude
Published: February 2012

The role of sympathetic innervation in the control of spontaneous fluctuations of cerebral blood flow is still poorly understood. In conscious, unrestrained rats, blood flow velocity (pulsed Doppler) was measured in both internal carotid arteries 1 week after either excision of the right superior cervical ganglion (n= 8) or sham surgery (n= 6). Using Fourier‐based techniques, spectral power of each carotid blood flow (CBF) was computed over the whole recording period (246 min), which was segmented into nine consecutive 27.3 min periods. Variability of CBF (spectral power) was ∼40% higher (P < 0.02) on the denervated than on the intact side at frequencies <1 Hz. Coherence between left and right CBFs was similar in the two groups of rats, except in the 0.01–0.1 Hz frequency range where it was lower (P < 0.05) in rats with unilateral sympathectomy (0.54 ± 0.03) than in intact rats (0.74 ± 0.06). In this frequency range, mathematically removing the influence of arterial pressure had little effect on coherence between CBFs in both groups of rats, so that coherence remained significantly lower in rats with unilateral sympathectomy (0.52 ± 0.03) than in intact rats (0.70 ± 0.06). This study indicates that sympathetic innervation has an overall buffering influence on CBF variability. This modulatory role is especially important in a frequency range corresponding to slow fluctuations of CBF (lasting from 10 to 100 s), which are essentially unrelated to fluctuations of arterial pressure.
Residual force enhancement after stretch in striated muscle. A consequence of increased myofilament overlap?
Authors: Edman K. A. P.
Published: February 2012

Abstract  When skeletal muscle is stretched above optimal sarcomere length during tetanic activity there is an increase in force that stays above the isometric force level throughout the activity period. This long‐lasting increase in contractile force, generally referred to as ‘residual force enhancement after stretch’ (FEresid), has been studied in great detail in various muscle preparations over more than half a century. Substantial evidence has been presented to show that non‐uniform sarcomere behaviour plays a major part in the development of FEresid. However, in a great number of recent studies the role of sarcomere non‐uniformity has been challenged and alternative mechanisms have instead been proposed to explain the increase in force such as enhancement of cross‐bridge function and/or strengthening of parallel elastic elements along the muscle fibres. This article presents a short review of the salient features of FEresid and provides evidence that non‐uniform sarcomere behaviour is indeed likely to play a major role in the development of FEresid. Electron microscopical studies of fibres rapidly fixed after active stretch demonstrate that, dispersed in the preparation, there are assymetrical length changes within the two halves of myofibrillar sarcomeres resulting in greater filament overlap in one half of the sarcomere than in the opposite sarcomere half. Sarcomere halves with increased filament overlap will consequently be in a situation where they are able to produce a greater force than that recorded in the isometric control. Weaker regions in series will be able to keep the enhanced force by recruitment of elastic elements.
The Effects of Fuel Characteristics on Stoichiometric Spark-Assisted HCCI
Authors: Weall Adam J. , Szybist James P.
Published: February 2012

The characteristics of fuel lean HCCI operation using a variety of fuels are well known and have been demonstrated using different engine concepts in the past. In contrast, stoichiometric operation of HCCI is less well documented. Recent studies have highlighted the benefits of operating at a stoichiometric condition in terms of load expansion combined with the applicability of three way catalyst technology to reduce NOx emissions. In this study the characterization of stoichiometric HCCI using gasoline-like fuels was undertaken. The fuels investigated are gasoline, a 50 vol% blend of iso-butanol and gasoline (IB50), and an 85% vol blend of ethanol and gasoline (E85). A single cylinder engine operating with direct injection and spark assist combined with a fully variable hydraulic valve actuation system allowed a wide range of operating parameters to be studied. This included the effects of negative valve overlap duration, intake valve closing and valve lift. Furthermore, the interaction between fuel injection timing and spark and how they can affect the required valve timing to achieve stoichiometric HCCI combustion are also studied. A comprehensive combustion and emissions analysis is conducted using gasoline, IB50 and E85 at an engine speed of 2000rpm over a range of operating loads. The resultant fuel properties which differed in terms of octane rating, fuel oxygenation and heat of vaporization show that stoichiometric HCCI is possible using a range of fuels but that these fuel characteristics do have some effect on the combustion characteristics. How these fuel properties can enable an increased engine operating envelope to be achieved, in comparison with both fuel lean HCCI and conventional spark ignited combustion, is then discussed.
Oil Consumption Measurements for a Modern Opposed-Piston Two-Stroke Diesel Engine
Authors: Callahan Brian J. , Wahl Michael H. , Froelund Kent
Published: February 2012

Opposed-piston two-stroke diesel engines have an inherent thermodynamic efficiency advantage and, by virtue of having double the firing frequency, allow for increased power density and lower NOX emissions while improving fuel efficiency, when compared to a four-stroke engine of equivalent power. However, opposed-piston two-stroke engines are piston-ported and, as such, are often erroneously dismissed for use in emissions compliant, on-highway vehicle applications over oil control concerns. The results presented in this paper show that oil control at levels acceptable for combustion and emissions control purposes is attainable with crankcase-lubricated, piston-ported opposed-piston diesel engines. Lubricant oil consumption was measured for the 13 test modes of the European Stationary Cycle using a real-time Da Vinci lubricant oil consumption measurement system. Repeatability of the measurement process was demonstrated. Oil consumption was measured during engine warm-up and shown to be reduced 30% compared to the fully warm condition. Furthermore, an increase of the oil control ring tension resulted in 16% lower oil consumption compared to the baseline. An optimization involving measurements with different cylinder kits resulted in a weighted average fuel-specific lubricant oil consumption of 0.18%. These data represent the first measured lubricant oil consumption maps for any contemporary two-stroke diesel engine ever reported.
Cylinder Liner in Ductile Cast Iron for High Loaded Combustion Diesel Engines
Authors: Rejowski Edney Deschauer , Soares Edmo , Roth Ingo , Rudolph Steffen
Published: February 2012

With the increase of combustion loading and the trend to reduce engine size, there is a need for thinner but stronger wet cylinder liners. While most of the current cylinder liners are made of gray cast iron, due to its good tribological behavior, machinability performance and competitive price, alternative casting materials like compact graphite iron, ductile iron and even steel are being considered to cover the future engine demands. In this paper, a new ductile iron (DI) cast material for wet cylinder liners is presented. The material has about 60 and 70% higher limits respectively for tensile stress and fatigue resistance as compared to conventional gray cast irons, but without penalty on the tribological properties. There is also a potential improvement to avoid cavitation on the outside surface due to its higher young modulus, which also equates to a higher stiffness. The tested cylinder liners were induction hardened on the running surface and a slide hone process was used to improve wear and scuffing resistance. The liners were tested in a HDD engine with PCP of 245 bar and showed similar wear as observed with conventional cylinder liners of gray cast iron material. The DI cylinder liners were also tested in an abusive scuffing engine test without any concern. The improved mechanical properties of the described new DI material introduce possibilities to reduce liner wall thickness or increase specific output. The preliminary evaluation in this paper showed that this new material is feasible for HDD diesel engines with PCP up to 250 bar. In cases that the customer needs to increase the bore diameter for output reasons there is the potential to reduce the liner wall thickness up to 25% based on high mechanical properties (UTS, Young Modulus and fatigue strength). In both cases, it’s recommended a FEA analysis to support the new component design.
Modeling of Lambda Sensor Output With Exhaust Gas Mixtures From Natural Gas-Fueled Engines
Authors: Toema Mohamed , Chapman Kirby S.
Published: February 2012

The increasingly strict emission regulations may require implementing Non-Selective Catalytic Reduction (NSCR) system as a promising emission control technology for stationary rich burn spark ignition engines. Many recent investigations used NSCR systems for stationary natural gas fueled engines showed that NSCR systems were unable to consistently control the emissions level below the compliance limits. Modeling of NSCR components to better understand, and then exploit, the underlying physical processes that occur in the lambda sensor and the catalyst media is now considered an essential step toward the required NSCR system performance. This paper presents the work done to date on a modeling of lambda sensor that provides feedback to the air-to-fuel controller. Several recent experimental studies indicate that the voltage signal from the lambda sensor may not be interpreted correctly because of the physical nature in the way the sensor senses the exhaust gas concentration. Correct interpretation of the sensor output signal is necessary to achieve consistently low emissions level. The goal of this modeling study is to improve the understanding of the physical processes that occur within the sensor, investigate the cross-sensitivity of various exhaust gas species on the sensor performance, and finally this model serves as a tool to improve NSCR control strategies. This model simulates the output from a planar switch type lambda sensor. The model consists of three modules. The first module models the multi-component mass transport through the sensor protective layer. Diffusion fluxes are calculated using the Maxwell-Stefan equation. The second module includes all the surface catalytic reactions that take place on the sensor platinum electrodes. All kinetic reactions are modeled based on the Langmuir-Hinshelwood kinetic mechanism. The model incorporates for the first time methane catalytic reactions on the sensor platinum electrode. The third module is responsible for simulating the reactions that occur on the electrolyte material and determine the sensor output voltage. The model results are validated using field test data obtained from a mapping study of a natural gas-fueled engine equipped with NSCR system. The data showed that the lambda sensor output voltage is influenced by the reducing species concentration, such as carbon monoxide (CO) and hydrogen (H2). The results from the developed model and the experimental data showed strong correlations between CO and H2 with the sensor output voltage within the lambda operating range between 0.994 to 1.007 (catalytic converter operating window). This model also showed that methane does not significantly influence the lambda sensor performance compared to the effect of CO and H2.
An “Inordinate Fondness for Transporters” Explained?
Authors: Eide David J.
Published: February 2012

The presence of low- and high-affinity transporters enables cells to adequately prepare for nutrient fluctuations.
Immune Sentinels
Authors: Mueller Kristen L.
Published: February 2012

A specialized population of B lymphocytes is important for controlling bacterial infections and preventing sepsis.
To Be or Not to Be
Authors: Wong Wei
Published: February 2012

By altering the transcriptional response to Notch signaling, the transcription factor Hamlet enables Notch to specify diverse cell fates.
Polyfunctional T Cells
Authors: Foley John F.
Published: February 2012

Temporal analysis of individual activated T cells reveals programs of sequential secretion of multiple effector cytokines.
Expression Turnover Profiling To Monitor the Antifungal Activities of Amphotericin B, Voriconazole, and Micafungin against Aspergillus fumigatus
Authors: Zhao Yanan , Paderu Padmaja , Park Steven , Dukhan Aleksandra , Senter Meredith , Perlin David S.
Published: February 2012

Eight highly expressed candidate genes were selected for mRNA profiling to monitor the transcriptome kinetics of
Aspergillus fumigatus
strains exposed to antifungal drugs as potential biomarkers of live cells to assess treatment efficacy. Mycelia were treated with fungicidal drugs amphotericin B and voriconazole, as well as the fungistatic drug micafungin. Transcription was monitored at 0, 4, 8, and 24 h posttreatment. The expression turnover profile provides a possible tool to assess antifungal therapy effects.
bla
NDM-1
Is a Chimera Likely Constructed in Acinetobacter baumannii
Authors: Toleman M. A. , Spencer J. , Jones L. , Walsh T. R.
Published: February 2012

Alignment of DNA sequences found upstream of
aphA6
and all
bla
NDM-1
genes displays 100% identity. This identity continues 19 bp into the
bla
NDM-1
gene such that the first 6 amino acids of
aphA6
and
bla
NDM-1
are the same. Furthermore, the percent GC content (GC%) of
aphA6
is considerably lower than that of
bla
NDM-1
and the GC% within the
bla
NDM-1
structural gene changes dramatically after the first 19 bp. This is unequivocal evidence that
bla
NDM-1
is a chimera.
Abrupt Temporal Fluctuations in the Chicken Fecal Microbiota Are Explained by Its Gastrointestinal Origin
Authors: Sekelja M. , Rud I. , Knutsen S. H. , Denstadli V. , Westereng B. , Næs T. , Rudi K.
Published: February 2012

One of the main challenges in understanding the composition of fecal microbiota is that it can consist of microbial mixtures originating from different gastrointestinal (GI) segments. Here, we addressed this challenge for broiler chicken feces using a direct 16S rRNA gene-sequencing approach combined with multivariate statistical analyses. Broiler feces were chosen because of easy sampling and the importance for pathogen transmission to the human food chain. Feces were sampled daily for 16 days from chickens with and without a feed structure-induced stimulation of the gastric barrier function. Overall, we found four dominant microbial phylogroups in the feces. Two of the phylogroups were related to clostridia, one to lactobacilli, and one to
Escherichia/Shigella
. The relative composition of these phylogroups showed apparent stochastic temporal fluctuations in feces. Analyses of dissected chickens at the end of the experiment, however, showed that the two clostridial phylogroups were correlated to the microbiota in the cecum/colon and the small intestine, while the upper gut (crop and gizzard) microbiota was correlated to the lactobacillus phylogroup. In addition, chickens with a stimulated gizzard also showed less of the proximate GI dominating bacterial group in the feces, supporting the importance of the gastric barrier function. In conclusion, our results suggest that GI origin is a main determinant for the chicken fecal microbiota composition. This knowledge will be important for future understanding of factors affecting shedding of both harmful and beneficial gastrointestinal bacteria through feces.
Equine Stomachs Harbor an Abundant and Diverse Mucosal Microbiota
Authors: Perkins G. A. , den Bakker H. C. , Burton A. J. , Erb H. N. , McDonough S. P. , McDonough P. L. , Parker J. , Rosenthal R. L. , Wiedmann M. , Dowd S. E. , Simpson K. W.
Published: February 2012

Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescencein situhybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 wereFirmicutes(>83% reads/sample), mainlyStreptococcusspp.,Lactobacillusspp. and,Sarcinaspp. Cluster 2 was more diverse, with predominantlyProteobacteria,Bacteroidetes, andFirmicutes, consisting ofActinobacillusspp.Moraxellaspp.,Prevotellaspp., andPorphyromonasspp.Helicobactersp. sequences were not identified in any of 53,920 reads. FISH (n= 9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly moreStreptococcusspp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual.
NAD(P)
+
-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N
2
Fixation Capabilities
Authors: Zhang Ye , Aono Toshihiro , Poole Phillip , Finan Turlough M.
Published: February 2012

C
4
-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N
2
-fixing bacteria (bacteroids) within legume nodules. In
Sinorhizobium meliloti
bacteroids from alfalfa, NAD
+
-malic enzyme (DME) is required for N
2
fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont
Rhizobium leguminosarum
, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that
dme
mutants of the broad-host-range
Sinorhizobium
sp. strain NGR234 formed nodules whose level of N
2
fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single
pckA
and single
dme
mutants fixed N
2
at reduced rates, a
pckA dme
double mutant had no N
2
-fixing activity (Fix
−
). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix
−
phenotype of
S. meliloti dme
mutants may be specific to the alfalfa-
S. meliloti
symbiosis. We therefore examined the ME-like genes
azc3656
and
azc0119
from
Azorhizobium caulinodans
, as
azc3656
mutants were previously shown to form Fix
−
nodules on the tropical legume
Sesbania rostrata
. We found that purified AZC3656 protein is an NAD(P)
+
-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N
2
fixation in
A. caulinodans
and
S. meliloti
, in other rhizobia this activity can be bypassed via another pathway(s).
Identification and Characterization of γ-Aminobutyric Acid Uptake System GabP

Cg

(NCgl0464) in Corynebacterium glutamicum
Authors: Zhao Zhi , Ding Jiu-Yuan , Ma Wen-hua , Zhou Ning-Yi , Liu Shuang-Jiang
Published: February 2012

Corynebacterium glutamicum
is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a
C. glutamicum
GABA-specific transporter (GabP

Cg

) encoded by
ncgl0464
was identified and characterized. GabP

Cg

plays a major role in GABA uptake and is essential to
C. glutamicum
growing on GABA. GABA uptake by GabP

Cg

was weakly competed by
l
-Asn and
l
-Gln and stimulated by sodium ion (Na
+
). The

K
m

and
V
max
values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min
−1
(mg dry weight [DW])
−1
, respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min
−1
(mg DW)
−1
, respectively, at pH 7.5. GabP

Cg

has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of
Escherichia coli
but low identities to the currently known GABA transporters (17% and 15% to
E. coli
GabP and
Bacillus subtilis
GabP, respectively). The mutant RES167 Δ
ncgl0464
/pGXKZ9 with the GabP

Cg

deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP

Cg

represents a new type of GABA transporter and is potentially important for engineering GABA-producing
C. glutamicum
strains.
Investigations of Salmonella enterica Serovar Newport Infections of Oysters by Using Immunohistochemistry and Knockout Mutagenesis
Authors: Morrison Christopher M. , Dial Sharon M. , Day William A. , Joens Lynn A.
Published: February 2012

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, withSalmonellabeing one of a number of pathogens that have been found in market oysters. Previous work by our lab found thatSalmonellawas capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigateSalmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show thatSalmonellawas capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function ofSalmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When theSalmonellapathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting thatSalmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.
Functional Assignment of YvgO, a Novel Set of Purified and Chemically Characterized Proteinaceous Antifungal Variants Produced by Bacillus thuringiensis SF361
Authors: Manns David C. , Churey John J. , Worobo Randy W.
Published: February 2012

This study reports a novel class of antifungal protein derived from bacterial origin.Bacillus thuringiensisSF361, the strain also responsible for producing the novel bacteriocin thurincin H, exhibits broad antifungal activity against select members of several fungal genera, includingAspergillus,Byssochlamys, andPenicillium, as well as the pathogenic yeastCandida albicans. Optimal antifungal production and secretion were observed after-log phase growth when incubated at 37°C in a carbohydrate-free growth medium. High-performance liquid chromatography purification was performed after pH-selective ammonium sulfate precipitation and size-exclusion chromatography. Intact mass analysis and peptide mass fingerprinting identified the 13,484-Da protein to be a mass homolog to the YvgO protein construct sequenced fromBacillus cereusAH 1134. Further analysis via amino-terminal sequencing also revealed the existence of four distinct yet equally efficacious YvgO variants differing only within the first four N-terminal residues. YvgO was found to be remarkably stable, maintaining its antifungal activity under a wide pH and temperature range. When assayed against the toxigenic speciesByssochlamys fulvaH25, the selected primary filamentous fungal indicator, the MIC was estimated to be 1.5 ppm.Candida albicans3153 was more resistant, exhibiting MICs between 25 and 800 ppm, depending on growth conditions. YvgO is unique among antifungals, showing no known sequential or functional homology to the typical classes of antifungal proteins, including common membrane-acting agents such as cellulases and glucanases. Due to its activity against an array of pathogenic and spoilage fungi, the potentials for clinical, agricultural, and food-processing applications are encouraging.
Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting the
groEL
Gene
Authors: Junick Jana , Blaut Michael
Published: February 2012

Quantitative real-time PCR assays targeting the
groEL
gene for the specific enumeration of 12 human fecal
Bifidobacterium
species were developed. The housekeeping gene
groEL
(
HSP60
in eukaryotes) was used as a discriminative marker for the differentiation of
Bifidobacterium adolescentis
,
B. angulatum
,
B. animalis
,
B. bifidum
,
B. breve
,
B. catenulatum
,
B. dentium
,
B. gallicum
,
B. longum
,
B. pseudocatenulatum
,
B. pseudolongum
, and
B. thermophilum
. The bifidobacterial chromosome contains a single copy of the
groEL
gene, allowing the determination of the cell number by quantification of the
groEL
copy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a given
Bifidobacterium
species. Independent of the
Bifidobacterium
species tested, the proportion of
groEL
copies recovered from fecal samples spiked with 5 to 9 log
10
cells/g feces was approximately 50%. The quantification limit was 5 to 6 log
10
groEL
copies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3
Bifidobacterium
species and
B. longum
frequently detected. The predominant species in infant and adult fecal samples were
B. breve
and
B. adolescentis
, respectively. It was possible to distinguish
B. catenulatum
and
B. pseudocatenulatum
. We conclude that the
groEL
gene is a suitable molecular marker for the specific and accurate quantification of human fecal
Bifidobacterium
species by real-time PCR.
Establishment of Characteristic Gut Bacteria during Development of the Honeybee Worker
Authors: Martinson Vincent G. , Moy Jamie , Moran Nancy A.
Published: February 2012

Previous surveys have shown that adult honeybee (Apis mellifera) workers harbor a characteristic gut microbiota that may play a significant role in bee health. For three major phylotypes within this microbiota, we have characterized distributions and abundances across the life cycle and among gut organs. These distinctive phylotypes, called Beta, Firm-5, and Gamma-1 (BFG), were assayed using quantitative PCR, fluorescentin situhybridization (FISH) microscopy, and the experimental manipulation of inoculation routes within developing bees. Adult workers (9 to 30 days posteclosion) contained a large BFG microbiota with a characteristic distribution among gut organs. The crop and midgut were nearly devoid of these phylotypes, while the ileum and rectum together contained more than 95% of the total BFG microbiota. The ileum contained a stratified community in which the Beta and Gamma-1 phylotypes dominated, filling the longitudinal folds of this organ. Deep sequencing of 16S rRNA genes showed clear differences among communities in midgut, ileum, and rectum. In contrast with older workers, larvae and newly emerged workers contain few or no bacteria, and their major food source, bee bread, lacks most characteristic phylotypes. In experiments aimed at determining the route of inoculation, newly emerged workers (NEWs) sometimes acquired the typical phylotypes through contact with older workers, contact with the hive, and emergence from the brood cell; however, transmission was patchy in these assays. Our results outline a colonization pattern for the characteristic phylotypes throughA. melliferaontogeny. We propose the names “CandidatusSnodgrassella alvi” and “CandidatusGilliamella apicola” for the Beta and Gamma-1 phylotypes, respectively.
Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins
Authors: Novoselova Tatiana V. , Zahira Kiran , Rose Ruth-Sarah , Sullivan James A.
Published: February 2012

Like other Nedd4 ligases,
Saccharomyces cerevisiae
E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of
BUL
gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using
in vitro
ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.
Streptococcus pneumoniae Can Utilize Multiple Sources of Hyaluronic Acid for Growth
Authors: Marion Carolyn , Stewart Jason M. , Tazi Mia F. , Burnaugh Amanda M. , Linke Caroline M. , Woodiga Shireen A. , King Samantha J.
Published: February 2012

The mechanisms by which
Streptococcus pneumoniae
obtains carbohydrates for growth during airway colonization remain to be elucidated. The low concentration of free carbohydrates in the normal human airway suggests that pneumococci must utilize complex glycan structures for growth. The glycosaminoglycan hyaluronic acid is present on the apical surface of airway epithelial cells. As pneumococci express a hyaluronate lyase (Hyl) that cleaves hyaluronic acid into disaccharides, we hypothesized that during colonization pneumococci utilize the released carbohydrates for growth. Hyaluronic acid supported significant pneumococcal growth in an
hyl
-dependent manner. A phosphoenolpyruvate-dependent phosphotransferase system (PTS) and an unsaturated glucuronyl hydrolase (Ugl) encoded downstream of
hyl
are also essential for growth on hyaluronic acid. This genomic arrangement is present in several other organisms, suggesting conservation of the utilization mechanism between species.
In vivo
experiments support the hypothesis that
S. pneumoniae
utilizes hyaluronic acid as a carbon source during colonization. We also demonstrate that pneumococci can utilize the hyaluronic acid capsule of other bacterial species for growth, suggesting an alternative carbohydrate source for pneumococcal growth. Together, these data support a novel function for pneumococcal degradation of hyaluronic acid
in vivo
and provide mechanistic details of growth on this glycosaminoglycan.
Signal Perception by the Secretion Stress-Responsive CssRS Two-Component System in Bacillus subtilis
Authors: Noone David , Botella Eric , Butler Clodagh , Hansen Annette , Jende Inga , Devine Kevin M.
Published: February 2012

The CssRS two-component system responds to heat and secretion stresses in
Bacillus subtilis
by controlling expression of HtrA and HtrB chaperone-type proteases and positively autoregulating its own expression. Here we report on the features of the CssS extracellular loop domain that are involved in signal perception and on CssS subcellular localization. Individual regions of the CssS extracellular loop domain contribute differently to signal perception and activation. The conserved hydrophilic 26-amino-acid segment juxtaposed to transmembrane helix 1 is involved in the switch between the deactivated and activated states, while the conserved 19-amino-acid hydrophobic segment juxtaposed to transmembrane 2 is required for signal perception and/or transduction. Perturbing the size of the extracellular loop domain increases CssS kinase activity and makes it unresponsive to secretion stress. CssS is localized primarily at the septum but is also found in a punctate pattern with lower intensity throughout the cell cylinder. Moreover, the CssRS-controlled HtrA and HtrB proteases are randomly distributed in foci throughout the cell surface, with more HtrB than HtrA foci in unstressed cells.