Genomic diversity of bacteriophages infecting Microbacterium spp.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Complete Genome Sequence of Rhodococcus erythropolis Phage Shuman.

Shuman is a bacteriophage isolated in Nyack, New York, using Rhodococcus erythropolis NRRL B-1574 as a host. It is a member of cluster CA and has a genome length of 46,544 bp. Shuman contains 67 predicted protein-coding genes, 3 tRNA genes, and no transfer-messenger RNA (tmRNA) genes.

Genome Sequences of Mycobacteriophages Findley, Hurricane, and TBond007.

We report here the genome sequences of three newly isolated phages that infect Mycobacterium smegmatis mc2155. Phages Findley, Hurricane, and TBond007 were discovered in geographically distinct locations and are related to cluster K mycobacteriophages, with Findley being similar to subcluster K2 phages and Hurricane and TBond007 being similar to subcluster K3 phages.

Lidocaine attenuates cognitive impairment after isoflurane anesthesia in old rats.

Post-operative cognitive dysfunction (POCD) is a clinical phenomenon that has drawn significant attention from the public and scientific community. Age is a risk factor for POCD. However, the contribution of general anesthesia/anesthetics to POCD and the underlying neuropathology are not clear. Here, we showed that 18-month-old male Fisher 344 rats exposed to 1.2% isoflurane, a general anesthetic, for 2h had significant learning and memory impairments assessed at 2-4 weeks after isoflurane exposure. These isoflurane effects were attenuated by intravenous lidocaine (1.5mg/kg as a bolus and then 2mg/kg/h during isoflurane exposure), a local anesthetic that has neuroprotective effect. Exposure to isoflurane or isoflurane plus lidocaine did not change the neuronal and synaptic density as well as the expression of NeuN (a neuronal protein), drebrin (a dendritic spine protein), synaptophysin (a synaptic protein), activated caspase 3 and caspase-activated DNase in the hippocampus at 29 days after isoflurane exposure when cognitive impairment was present. Isoflurane and lidocaine did not affect the amount of ß-amyloid peptide, total tau and phospho-tau in the cerebral cortex as well as interleukin-1ß and tumor necrosis factor-α in the hippocampus at 29 days after isoflurane exposure. Thus, isoflurane induces learning and memory impairment in old rats. Lidocaine attenuates these isoflurane effects. Isoflurane may not cause long-lasting neuropathological changes.

Inhibition of isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3 by serine 465 sequence-specific peptides.

Excitatory amino acid transporters (EAAT) transport glutamate into cells to regulate glutamate neurotransmission and to maintain nontoxic extracellular glutamate levels for neurons. We showed previously that the commonly used volatile anesthetic isoflurane increases the transporting activity of EAAT3, the major neuronal EAAT. This effect requires a protein kinase C (PKC) α-mediated and S465-dependent EAAT3 redistribution to the plasma membrane. Thus, we hypothesize that specific peptides can be designed to block this effect. We conjugated a 10-amino acid synthetic peptide with a sequence identical to that of EAAT3 around the S465 to a peptide that can facilitate permeation of the plasma membrane. This fusion peptide inhibited the isoflurane-increased EAAT3 activity and redistribution to the plasma membrane in C6 cells and hippocampus. It did not affect the basal EAAT3 activity. This peptide also attenuated isoflurane-induced increase of PKCα in the immunoprecipitates produced by an anti-EAAT3 antibody. A scrambled peptide that has the same amino acid composition as the S465 sequence-specific peptide but has a random sequence did not change the effects of isoflurane on EAAT3. The S465 sequence-specific peptide, but not the scrambled peptide, is a good PKCα substrate in in vitro assay. These peptides did not affect cell viability. These results, along with our previous findings, strongly suggest that PKCα interacts with EAAT3 to regulate its functions. The S465 sequence-specific peptide may interrupt this interaction and is an effective inhibitor for the regulation of EAAT3 activity and trafficking by PKCα and isoflurane.

Critical role of s465 in protein kinase C-increased rat glutamate transporter type 3 activity.

Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes.

OBJECTIVES: Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain. METHODS: EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of L-glutamate in the presence or absence of 0.64 microM amitriptyline. KEY FINDINGS: Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38-1.27 microM amitriptyline. Amitriptyline 0.64 microM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for L-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 microM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate. CONCLUSIONS: Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neurotransmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Inability of volatile anesthetics to inhibit oxygen-glucose deprivation-induced glutamate release via glutamate transporters and anion channels in rat corticostriatal slices.

Ischemia-induced extracellular glutamate accumulation and the subsequent excitotoxicity contribute significantly to ischemic brain injury. Volatile anesthetics have been shown to reduce ischemic brain injury. Here, we showed that oxygen-glucose deprivation (OGD, to simulate ischemia in vitro) increased extracellular glutamate accumulation in the corticostriatal slices of adult rats. This increased accumulation was reduced by dihydrokinate, a glutamate transporter type 2 inhibitor, and 4,4'-dinitrostilbene-2,2'-disulfonic acid, a blocker for volume-activated anion channels. The volatile anesthetics isoflurane, sevoflurane and desflurane at clinically relevant concentrations did not affect the OGD-induced extracellular glutamate accumulation from brain slices of adult rats. Isoflurane also did not change the OGD-induced extracellular glutamate accumulation from brain slices of newborn/young rats. These results suggest that the OGD-induced glutamate accumulation involves reversed transport of glutamate via glutamate transporters and volume-activated anion channels. Volatile anesthetics may not inhibit this extracellular glutamate accumulation.

Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

Effects of volatile anesthetics on glutamate transporter, excitatory amino acid transporter type 3: the role of protein kinase C.

BACKGROUND: Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. The authors studied the effects of volatile anesthetics on one type of glutamate transporters, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C in mediating these effects. METHODS: Excitatory amino acid transporter type 3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Using two-electrode voltage clamp, membrane currents were recorded before, during, and after application of L-glutamate. Responses were quantified by integrating the current trace and are reported as microcoulombs. Data are mean +/- SEM. RESULTS: L-Glutamate-induced responses were increased gradually with the increased concentrations of isoflurane, a volatile anesthetic. At 0.52 and 0.70 mm isoflurane, the inward current was significantly increased compared with control. Isoflurane (0.70 mm) significantly increased Vmax (maximum velocity) (3.6 +/- 0.4 to 5.1 +/- 0.4 microC; P < 0.05) but not Km (Michoelis-Menten Constant) (55.4 +/- 17.0 vs. 61.7 +/- 13.6 microm; P > 0.05) of EAAT3 for glutamate compared with control. Treatment of the oocytes with phorbol-12-myrisate-13-acetate, a protein kinase C activator, caused a significant increase in transporter current (1.7 +/- 0.2 to 2.5 +/- 0.2 microC; P < 0.05). Responses in the presence of the combination of phorbol-12-myrisate-13-acetate and volatile anesthetics (isoflurane, halothane, or sevoflurane) were not greater than those when volatile anesthetic was present alone. Oocytes pretreated with any of the three protein kinase C inhibitors alone (chelerythrine, staurosporine, or calphostin C) did not affect basal transporter current. Although chelerythrine did not change the anesthetic effects on the activity of EAAT3, staurosporine or calphostin C abolished the anesthetic-induced increase of EAAT3 activity. CONCLUSIONS: These data suggest that volatile anesthetics enhance EAAT3 activity and that protein kinase C is involved in mediating these anesthetic effects.