“Memory system circuitry may regulate how cues associated


“Memory system circuitry may regulate how cues associated check details with cocaine are extinguished, and understanding neurosubstrates of extinction may lead to the development of improved treatment strategies for cocaine addiction. Sites

within the hippocampus and amygdala were investigated for their role in regulating cocaine cue extinction learning. Initially, rats were trained to self-administer cocaine under a second-order reinforcement schedule (cocaine and cocaine cues present) followed by a 2-week abstinence period. Using lidocaine, rats next underwent bilateral inactivation of the dorsal subiculum (dSUB) or rostral basolateral amygdala (rBLA), asymmetric inactivation of the dSUB and rBLA, unilateral inactivation of the dSUB or rBLA, or ipsilateral inactivation of the dSUB and rBLA prior to cocaine

cue extinction training sessions (only cocaine cues present) on two consecutive days. Relative to vehicle, bilateral and asymmetric lidocaine treatments in the dSUB and rBLA slowed cocaine cue extinction learning. Specifically, vehicle-treated rats exhibited a significantly larger difference in responding from buy Saracatinib Day 1 to Day 2 of extinction training than lidocaine-treated rats. In comparison, unilateral or ipsilateral lidocaine treatments in the dSUB and rBLA did not slow cocaine cue extinction learning. Rats treated with lidocaine and vehicle exhibited a similar difference in responding from Day 1 to Day 2 of extinction training. These results indicate that sites within the hippocampus and amygdala need to be functionally active simultaneously in at least one brain hemisphere for acquisition of cocaine cue extinction learning. These results further suggest that a serial circuit within each hemisphere mediates acquisition of cocaine cue extinction learning. “
“Giant cells of the cochlear nucleus are thought to integrate multimodal Morin Hydrate sensory inputs and participate in monaural sound

source localization. Our aim was to explore the significance of a hyperpolarization-activated current in determining the activity of giant neurones in slices prepared from 10 to 14-day-old rats. When subjected to hyperpolarizing stimuli, giant cells produced a 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288)-sensitive inward current with a reversal potential and half-activation voltage of –36 and –88 mV, respectively. Consequently, the current was identified as the hyperpolarization-activated non-specific cationic current (Ih). At the resting membrane potential, 3.5% of the maximum Ih conductance was available. Immunohistochemistry experiments suggested that hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective (HCN)1, HCN2, and HCN4 subunits contribute to the assembly of the functional channels. Inhibition of Ih hyperpolarized the membrane by 6 mV and impeded spontaneous firing.

The video contained 300 frames and each frame was presented to th

The video contained 300 frames and each frame was presented to the model for 40 ms of simulation time. Each image was originally 256 × 256 pixels. Because our cortical model is made up of single columns, however, the input size was reduced to Staurosporine cost 20 × 20 pixels (see Fig. 2B) to approximate the visual space that would drive neurons in a receptive field of a V1 cortical column. This was an assumed approximation given the 100 deg2 receptive field and 36 × 36 (64 × 64 pixel) input from the Goard and Dan experiment.

In the 256 × 256 pixel image, RF1 received input from pixels (121–140) × (121–140) and RF2 received input from pixels (141–160) × (121–140). Figure 3 shows the architecture of RF1 and RF2. It has been shown that retinal neurons remove linear correlations by ‘whitening’ images before they reach the cortex (Simoncelli & Olshausen, 2001). To simulate this, all the images were whitened and normalised before being presented to the network (Fig. 2B). Whitening was achieved by applying a Gaussian filter to the Fourier-transformed image (see http://redwood.berkeley.edu/bruno/npb261b/). This flattens the power spectrum of the image Ensartinib supplier and is essentially equivalent to convolving the image with an on-center off-surround filter, as is observed in retinal

ganglion cells and the lateral geniculate nucleus (LGN). As we were not interested in modeling orientation selectivity development, we assumed that the simulated V1 columns, RF1 and RF2, were selective to vertical edges. Therefore, the images were convolved with a vertical Gabor filter after whitening.

The Gabor filter was constructed by modulating a Gabor kernel with a sinusoidal wave as shown in Eqn. (1), where σx and σy determine the spatial extent of the Gaussian in x and y and f specifies the preferred spatial wavelength Palmatine (Dayan & Abbott, 2001). Excitatory Poisson spike generators converted the images into spike trains in the input layer. (1) To develop our model, we used a publicly available simulator, which has been shown to simulate large-scale spiking neural networks efficiently and flexibly (Richert et al., 2011). The model contained a TRN, LGN, BF, two prefrontal cortex areas (providing top-down attention) and two, four-layered cortical microcircuits (Fig. 3). The cortical microcircuit architecture was adapted from Wagatsuma et al. (2011), which was able to account for experimental observations of attentional effects on visual neuronal responses and showed that top-down signals enhanced responses in layers 2/3 and 5. All connections that occur between layers in a microcircuit are shown in Fig. 3. Within each layer, there are excitatory–excitatory, excitatory–inhibitory, inhibitory-excitatory and inhibitory–inhibitory connections (data not shown). Connection probabilities in our cortical model were the same as used in Wagatsuma et al. (2011) and are given in Table 1. All subcortical and top-down connection probabilities were set to 0.

citrinum (69% identity) and P putida UW4 (54% identity) The con

citrinum (69% identity) and P. putida UW4 (54% identity). The conserved glutamate (Glu) and leucine (Leu) amino acid residues that distinguish ACCDs (at the position of Glu295 and Leu322 in P. putida UW4) are marked with a box. Comparison of the ACCD sequence of T. asperellum with other two efficient biocontrol and plant growth promoters Trichoderma spp., T. virens and T. atroviride, whose genomes are now available, shows 91% and 94% identities at the protein level, respectively. At a nucleotide level, 85% and 89% identities are found, respectively. All the three genes have a small intron (55–71 bp) in a conserved position. The ACCD average

Ceritinib manufacturer activity of T. asperellum in submerged cultures with ACC as the sole nitrogen source was found to be 12.16±3.8 μmol α-ketobutyrate mg−1 protein h−1. An average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR (Fig. 2a) after 24 h of growth. No significant differences in activity could be detected after induction with different amounts of ACC tested (0.3–3 mM). Coculture with cucumber roots revealed in quantitative RT-PCR analysis a 1.8-fold induction of the gene that was no longer detectable after 12 h (data not shown), and no detectable protein activity was measured in these samples. Heterologous expression in E. check details coli under the inducible tac promoter was assayed in five different clones and the average activity was estimated to be

1500±380 nmol α-ketobutyrate mg−1 protein h−1. No significant differences in activity could be detected at all the tested IPTG concentrations (0.1–1 mM). Very low activity could be detected in noninduced clones (Table 1). A clone was chosen for a growth promotion assay and a significant (P<0.05) increase in root length, comparable with that induced by P. putida UW4, could be measured (Table 1). Tas-acdS RNAi transformants were obtained and subcultured to mitotic stability by repeated transfer on selective medium. Inhibition of Tas-acdS expression was followed by quantitative RT-PCR on mRNA extracted from cultures grown in ACC induction medium for 24 h. Various degrees of inhibition

could be detected in the different transformants (Fig. 2a). Clones #2 and #3, which presented growth rates and sporulation similar to the wild type on SM and that exhibited 95% reduction in mRNA expression (Fig. stiripentol 2a), were selected and evaluated for enzyme activity and root growth promotion. As shown in Fig. 2b, the two transformants had no detectable ACCD activity when grown on ACC as the sole nitrogen source, whereas activity could be measured in the induced wild type (WTi). Also, these two transformants could not grow on solid SM supplied with ACC as the nitrogen source (data not shown). Figure 3a presents the typical data obtained in one out of three independent pouch growth assays. Seed treatment with Trichoderma wild-type spores induced a significant (P<0.05) growth response in the seedlings.

Further analysis of clinical data and studies involving additiona

Further analysis of clinical data and studies involving additional sets of patients for verification of this hypothesis will provide a clearer picture helping to link genetic features with evidence-led clinical management of P. aeruginosa keratitis. The Microbiology Ophthalmic Group (MOG) includes Stephen Tuft, Stephen Kaye, Timothy Neal, Derek Tole, John Leeming, Peter McDonnell, Francisco Figueiredo, Fiona Carley, Malcolm Armstrong, Colin WIlloughby, Johnny Moore, Grace Ong. This work was supported by the UKNIHR

and a Dr Hans and Mrs Gertrude Hirsch Awards Scheme Fight for Sight Small Projects Grant. S.T. is supported by the NIHR Biomedical Research Centre for Ophthalmology, Moorfields GSK-3 beta pathway Eye Hospital. J.S. and H.S. contributed equally to this work. “
“Nhe (‘nonhaemolytic enterotoxin’) is a three-component cytotoxin implicated in the pathogenesis of diarrhoea Selleckchem Temozolomide by Bacillus

cereus. Nhe forms pores in pure lipid bilayers, but the function of the individual components (NheA,NheB and NheC) remains unclear. NheB and NheC are structural homologues of ClyA, a pore-forming cytotoxin of Escherichia coli. The non-ionic detergent dodecyl maltoside (DDM) has been shown to inhibit haemolysis of ClyA. We used DDM as a probe to examine the response of the Nhe proteins to DDM micelles. At its critical micellar concentration (0.2 mM), DDM inhibited propidium uptake by the native Nhe complex in Vero and HT29 cell suspensions. Pre-incubation of NheC with DDM did not inhibit cytotoxicity. NheB exhibited marked changes in 1-anilinonaphthalene-8-sulphonic acid (ANS) fluorescence after pre-exposure to DDM. Pre-incubation of NheB with DDM resulted in large molecular weight complexes as detected by size exclusion chromatography and diffusion through sized dialysis membranes and prevented binding of NheB to Vero cell monolayers. These data support a model in which conformational changes and oligomerization of NheB are prerequisite events in the 2-hydroxyphytanoyl-CoA lyase process of pore formation. The mechanisms by which Bacillus cereus causes diarrhoea in man remain unknown. Two of the putative enterotoxins, haemolysin BL (HBl) and Nhe (see Stenfors Arnesen et al., 2008), are three-component

cytotoxins. Nhe is cytolytic against both erythrocytes and epithelia because of osmotic lysis induced by pores formed in the host cell plasma membrane (Fagerlund et al., 2008). In epithelial cells, all three Nhe components are necessary for maximal cytotoxic activity (Lund & Granum, 1997). However, in certain cell types, namely rat pituitary GH4 cells, only NheA and NheB are necessary for pore formation and cytotoxicity (Haug et al., 2010). NheB and NheC have significant amino acid sequence homology to the HBl proteins. Using the crystal structure of HBl-B as the template, homology modelling predicts that two of the Nhe components, NheB and NheC, possess marked structural resemblance to ClyA of Escherichia coli (Fagerlund et al., 2008).

The transient nonchemotactic phenotype of V cholerae shed in sto

The transient nonchemotactic phenotype of V. cholerae shed in stool enhances infectivity within the next host (Merrell et al., 2002), by allowing the organisms to colonize

regions of the upper intestine not colonized by laboratory-grown bacteria. This suggests that chemotaxis leads the bacteria to the lower small intestine. AcfB and TcpI, by virtue of being positively regulated by ToxT, are specifically expressed inside the host intestine, and because the loss of both leads to lower levels of intestinal colonization, we suggest that these MCPs contribute to chemotaxis toward the correct intestinal location. However, the acfB tcpI check details mutant colonized the distal end of the intestine, similar to the wild-type strain, albeit at lower levels, and so these MCPs may be involved in localization CP-673451 datasheet within the microenvironment of the ileum. MCPs can function by either binding a chemoattractant, which facilitates swimming toward a gradient, or a chemorepellant, which facilitates swimming away from a gradient. We propose that AcfB and TcpI either recognize attractants within the intestinal crypts in the distal ileum or repellants present within the intestinal lumen at this location.

Upon acquisition of the VPI, V. cholerae gains the colonization factor and the regulatory elements to allow this organism to successfully colonize the intestine in response to the environmental conditions present there. The contribution of MCPs located within the VPI to intestinal colonization suggests that the VPI

also contains the ‘road map’ that directs the bacteria to the appropriate location to colonize. We thank Andy Camilli for providing plasmids. This work was supported by AI 43486 to K.E.K. Fig. S1. ClustalW alignment of AcfB and TcpI. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that Amisulpride aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs < 130 μM). The peptide adopted high levels of membrane-interactive α-helical structure (> 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m−1) and lyse them (> 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents. “
“A dimeric cytochrome c with an apparent molecular mass of 25 kDa was isolated from an anammox bacterium, strain KSU-1, in a relatively large quantity. This protein was named the NaxLS complex.

These results are consistent with the reduced levels of hippocamp

These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a

diet-specific click here manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets. “
“The Rehabilitation Gaming System (RGS) has been designed as a flexible, virtual-reality (VR)-based device for rehabilitation of neurological patients. Recently, training of visuomotor processing with the RGS was shown to effectively improve arm function in acute and chronic stroke patients. It is assumed that the VR-based training protocol related to RGS creates conditions that aid recovery by virtue of the human mirror neuron system. Here, we provide evidence for this assumption by identifying the brain areas involved in controlling the catching of approaching colored balls in the virtual buy AZD1208 environment of the RGS. We used functional magnetic resonance imaging of 18 right-handed healthy subjects (24 ± 3 years) in both active and imagination conditions. We observed that the imagery

of target catching was related to activation of frontal, parietal, temporal, cingulate and cerebellar regions. We interpret these activations in relation to object processing, attention, mirror mechanisms, and motor intention. Active catching followed an anticipatory mode, and resulted in significantly less activity in the motor control areas. Our results provide preliminary support for the hypothesis underlying RGS that this novel neurorehabilitation approach engages human mirror mechanisms that can be employed for visuomotor training. Rehabilitation of neurological patients is a major challenge. Given that

stroke is a primary cause of permanent disability (Mukherjee & Patil, 2011), there is a wide demand for rehabilitation of neurological deficits after stroke. Neurological deficits resulting from stroke differ in severity, owing to different Ribose-5-phosphate isomerase lesion locations, lesion volumes, and times elapsed since stroke (Seitz & Donnan, 2010). In this regard, a training program of basic arm–hand functions has been developed that scales in difficulty relative to the severity of the individual stroke survivor’s deficit on a session-by-session basis (Platz et al., 2009). Furthermore, it is well established that a dosing effect associated with more intense rehabilitative training leads to better neurological outcomes (Hummelsheim et al., 1995; Kwakkel et al., 1999).

All mutants constructed were verified by qRT-PCR, and all pnp mut

All mutants constructed were verified by qRT-PCR, and all pnp mutations were further verified by immunoblotting. Immunoblotting of SDS–PAGE gels was performed as outlined in QIAexpress® Detection Assay Handbook (Qiagen) using polyvinylidene difluoride membranes (HybondTM P; Amersham). A polyclonal rabbit anti-PNPase serum, generated by immunizing rabbits with purified PNPase (Clements et al., 2002) under

the ethical permit Dnr 79/2001, was used as primary antibody (1 : 500), whilst horseradish peroxidase–conjugated goat anti-rabbit-IgG (1 : 5000, Pierce) was applied as secondary antibody. Blots were analysed using SuperSignal detection kit (Pierce) and ChemiDoc XRS system (Bio-Rad). see more For extraction of RNA, bacteria were grown in LB to the exponential phase of growth. Total bacterial RNA was isolated using the TRI Reagent (Sigma). To determine transcripts spanning the pnp–nlpI and nlpI–deaD genes, RNA samples were first reverse-transcribed using the M-MLV kit (Invitrogen).

Standard PCR was then carried out on the cDNA products using different sets of primers (Supporting Information, Table S1). Total genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega) and used as a positive template control. DNA fragments were analysed on ethidium bromide–stained agarose gels. RNA quantification was carried out by quantitative reverse PCR using the SYBR Green JumpStart kit (Sigma) and the ABI Prism 7000 sequence detection system with primers detailed in Table S1. The ability to sustain sudden drop in growth temperature was assayed in two ways. First, bacteria were grown in LB to GSK126 nmr the exponential phase of growth at 37 °C. From that, Aurora Kinase 100 μL of the culture was adjusted to an OD600 nm of 0.5, which then was inoculated into 50 mL of precooled LB on a shaker at 15 °C. Optical densities of the cultures were then followed at regular time intervals. In the second assay, bacteria were grown

over night in LB at 37 °C and diluted in phosphate-buffered saline to an OD600 nm of 0.5. Subsequent 1 : 10 serial dilutions were then inoculated in 10 μL drops on two separate LB agar plates. One was incubated at 37 °C and the other at 15 °C. In S. Typhimurium, pnp and nlpI are linked and read from the same DNA strand (McClelland et al., 2001). The pnp STOP codon and the nlpI START codons are separated by 109 nucleotides (McClelland et al., 2001; Fig. 1a). Because of the close proximity of pnp to nlpI, we examined to what extent mutations in pnp would affect expression of nlpI. We compared the pnp and nlpI mRNA levels in the wild-type S. Typhimurium SR-11 (MC1) and three pnp mutants. These mutants were MC71 (pnp−) expressing a truncated PNPase because of a point mutation replacing codon 600 with stop codon TAA and mutant SFR228 (∆pnp) having the entire pnp open reading frame deleted (Fig. 1a) (Clements et al., 2002; Rouf et al., 2011).

All mutants constructed were verified by qRT-PCR, and all pnp mut

All mutants constructed were verified by qRT-PCR, and all pnp mutations were further verified by immunoblotting. Immunoblotting of SDS–PAGE gels was performed as outlined in QIAexpress® Detection Assay Handbook (Qiagen) using polyvinylidene difluoride membranes (HybondTM P; Amersham). A polyclonal rabbit anti-PNPase serum, generated by immunizing rabbits with purified PNPase (Clements et al., 2002) under

the ethical permit Dnr 79/2001, was used as primary antibody (1 : 500), whilst horseradish peroxidase–conjugated goat anti-rabbit-IgG (1 : 5000, Pierce) was applied as secondary antibody. Blots were analysed using SuperSignal detection kit (Pierce) and ChemiDoc XRS system (Bio-Rad). DAPT For extraction of RNA, bacteria were grown in LB to the exponential phase of growth. Total bacterial RNA was isolated using the TRI Reagent (Sigma). To determine transcripts spanning the pnp–nlpI and nlpI–deaD genes, RNA samples were first reverse-transcribed using the M-MLV kit (Invitrogen).

Standard PCR was then carried out on the cDNA products using different sets of primers (Supporting Information, Table S1). Total genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega) and used as a positive template control. DNA fragments were analysed on ethidium bromide–stained agarose gels. RNA quantification was carried out by quantitative reverse PCR using the SYBR Green JumpStart kit (Sigma) and the ABI Prism 7000 sequence detection system with primers detailed in Table S1. The ability to sustain sudden drop in growth temperature was assayed in two ways. First, bacteria were grown in LB to Luminespib price the exponential phase of growth at 37 °C. From that, Roflumilast 100 μL of the culture was adjusted to an OD600 nm of 0.5, which then was inoculated into 50 mL of precooled LB on a shaker at 15 °C. Optical densities of the cultures were then followed at regular time intervals. In the second assay, bacteria were grown

over night in LB at 37 °C and diluted in phosphate-buffered saline to an OD600 nm of 0.5. Subsequent 1 : 10 serial dilutions were then inoculated in 10 μL drops on two separate LB agar plates. One was incubated at 37 °C and the other at 15 °C. In S. Typhimurium, pnp and nlpI are linked and read from the same DNA strand (McClelland et al., 2001). The pnp STOP codon and the nlpI START codons are separated by 109 nucleotides (McClelland et al., 2001; Fig. 1a). Because of the close proximity of pnp to nlpI, we examined to what extent mutations in pnp would affect expression of nlpI. We compared the pnp and nlpI mRNA levels in the wild-type S. Typhimurium SR-11 (MC1) and three pnp mutants. These mutants were MC71 (pnp−) expressing a truncated PNPase because of a point mutation replacing codon 600 with stop codon TAA and mutant SFR228 (∆pnp) having the entire pnp open reading frame deleted (Fig. 1a) (Clements et al., 2002; Rouf et al., 2011).

94–099; P < 005) and elevated urinary microalbumin (OR 102; 95

94–0.99; P < 0.05) and elevated urinary microalbumin (OR 1.02; 95% CI 1.01–1.03; P < 0.05) were significantly associated with anti-diabetic medication treatment. The only independent factor associated with pharmacological treatment for hypertension was elevated HbA1c (OR 1.4; 95% CI 1.0–2.0; P < 0.05). Patient factors associated with prescription of lipid-lowering agents

were a past history of cardiovascular disease (OR 5.0; 95% CI 2.0–12.5; P < 0.001), GSK1120212 order concurrent use of anti-hypertensive agents (OR 2.6; 95% CI 1.2–5.8; P < 0.05) and elevated triglyceride (OR 1.9; 95% CI 1.2–3.1; P < 0.01). Treatment targets were not being translated into clinical practice in this cohort of patients with type 2 diabetes. Patients with acceptable HbA1c levels, with no history of cardiovascular disease and those taking few medications were at risk of being overlooked for the pharmacotherapy they

required. “
“Objective The purpose of this study is to examine the unit costs of a multi-service hospital in Palestine for the period 2005–2007. We investigate the cost structure of the Rafidya Hospital located in Nablus city, FK228 cost for both inpatient and outpatient departments. Methods This study uses cost–volume–profit (CVP) analysis, also known as breakeven analysis. CVP analysis requires examining total costs, along with fixed and variable costs. CVP analysis illuminates how changes in assumptions about cost behaviour and the relevant range in which those assumptions are valid affect the relationships among revenues, variable costs and fixed costs at various production levels. Key findings For the hospital of interest, we find that fixed costs account for 70% of total costs, and variable costs were 30% of total costs. Inpatient departments accounted for 86% of total costs, and outpatient departments were 14% of total costs. Results of the breakeven analysis illustrate that several departments charge sufficient fees to cover all unit costs. Conclusions Results provide useful information about unit cost based on four categories: (1) unit cost per admission of each department, (2) unit cost per patient day of each department, (3) unit cost per admission with

annual capital cost of each department and (4) unit cost per patient day with annual capital cost. Our results provide hospital cost information that can be used Farnesyltransferase by decision-makers to provide and expand healthcare services, in an effort to increase sustainability and profitability. The use of cost analysis by administrators and regulators will improve the quality of financial information, as well as enhance the efficient use of scarce resources. “
“Mortality and morbidity are increased in patients experiencing drug–drug interactions (DDIs). Critically ill patients are at an increased risk of adverse events from DDIs due to the large number of medications that they take and their changes in organ function. Currently, there is a lack of literature describing DDIs in the intensive care unit (ICU).

For quantitative analysis, tridecanoic acid was used as an intern

For quantitative analysis, tridecanoic acid was used as an internal standard. All determinations were performed in triplicate experiments. The data were recorded as means and SDs. The polysaccharide was isolated from freeze-dried cells using the classical alkali treatment as has been reported previously

(Elbein & Mitchell, 1973; Gunja-Smith et al., 1977; Lillie & Pringle, 1980; Lou et al., 1997), and visualized using a TLC method (Seibold & Eikmanns, 2007). Total CYC202 concentration polysaccharide was determined using the phenol–sulfuric acid method (Dubois et al., 1956). For quantitative analysis, the extracted polysaccharide was digested with α-amylase (; 10 IU mg−1 of dried polysaccharide; Sigma) and amyloglucosidase (; 20 IU mg−1 of dried polysaccharide; Sigma) in 50 mM sodium acetate buffer (pH 5) at 55 °C for 3–4 h and with gentle vortexing. Commercial glycogen standard (1 mg mL−1) was used as a control

for enzymatic hydrolysis. The amount of glucose formed under these conditions was taken as a measure of glycogen in cells. Glucose was determined using a specific glucose oxidase method (Keston, 1956). All determinations were performed in triplicate experiments. The data were recorded Navitoclax chemical structure as means and SDs. Various genomic databases of Rhodococcus strains are now available for public research. Among them, the genome sequence of R. jostii strain RHA1 has been the first sequence publicly available for the screening and identification of genes and metabolic pathways (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). Recently, we identified six putative genes (glgA, glgB, glgC, glgE, glgP and glgX) involved in glycogen biosynthesis and mobilization in a genome-wide bioinformatic study of the genomic database Montelukast Sodium of strain RHA1 (Hernández et al., 2008). Using these RHA1 sequences, we performed a genome-wide examination of key genes involved in glycogen metabolism in the available databases of R. opacus B4, Rhodococcus erythropolis PR4 and R. erythropolis SK121. The degree of identity of full protein sequences of these species is shown in Table 2. In all cases, a high identity between orthologous

proteins was observed. In general, we observed similar gene arrangements in all strains, with little differences. The glgB, glgE and glgP genes occurred in a cluster, whereas glgA and glgC were adjacent and clustered in the opposite orientation. Only in the R. erythropolis SK121 genome was a gene coding for a putative O-methyltransferase enzyme found between glgA and glgC. Finally, glgX was located in a separate cluster associated with another carbohydrate metabolism gene, which encodes a putative 1–4-α-d-glucan 1-α-d-glucosylmutase (also called maltooligosyl trehalose synthase) in the genome of all the strains studied. These results suggested that the different strains possess the genetic potential to synthesize and mobilize glycogen.