Biological destruction of CCl(4): I. Experimental design and data

A denitrifying consortium capable of transforming carbon tetrachloride (CCl(4)) was cultured from aquifer sediment from the U.S. Department of Energy's Hanford Site in southeastern Washington State. To understand the kinetics of the biological destruction of CCl(4) by these microbes, a set of experiments, the conditions of which were chosen according to a fractional factorial experimental design, were completed. This article reports on the experimental design along with the results for CCl(4), biomass, acetate, nitrate, and nitrite concentrations. These data indicate that growth is inhibited by high nitrite concentrations, whereas CCl(4) degradation is slowed by the presence of nitrate and/or nitrite. (c) 1994 John Wiley & Sons, Inc.     

Biotransformation of carbon tetrachloride by various acetate‐ and nitrate‐limited denitrifying consortia

Fed‐batch experiments were performed to determine the carbon tetrachloride (CT)‐degrading ability of three denitrifying consortia cultured from sites not contaminated with CT. A mathematical model was used to quantify the rates of CT transformation by the consortia under both acetate‐limiting and nitrate‐limiting conditions. A rate constant for CT transformation on a cellular protein basis and the fraction of degraded CT transformed to chloroform (CF) were determined for each consortium by optimizing the model to fit the experimental data. The parameters for these experiments were statistically compared to those obtained for previous experiments with a denitrifying consortium cultured from an aquifer soil sample from the US Department of Energy Hanford site in southeastern Washington state. Results of F‐test analysis indicated the rate of CT transformation and the production of CF both were functions of the limiting nutrient. Under nitrate‐limited conditions, the rate constant for CT transformation for all four consortia was about 30 L/gmol/min and approximately 50% of the CT transformed was converted to CF. When acetate was the limiting nutrient, the rate constant for CT transformation was approximately 8 L/gmol/min and the CF yield decreased to about 25%. These results imply the ability to degrade CT may be inherent to some denitrifying organisms, regardless of previous exposure to CT. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 342–348, 1999.     

PI3K/Akt Pathway is Required for Spinal Central Sensitization in Neuropathic Pain

Phosphatidylinositol-3-kinase (PI3K) has been identified in the expression of central sensitization after noxious inflammatory stimuli. However, its contribution in neuropathic pain remains to be determined. Here we address the role of PI3K signaling in central sensitization in a model of neuropathic pain, and propose a novel potential drug target for neuropathic pain. Chronic constriction injury (CCI) rat model was used in the study as the model for neuropathic pain. Western blotting, whole-cell patch clamp, and von Frey assay were performed to study biochemical, electrical, and behavioral changes in CCI rats, respectively. A steroid metabolite of the fungi (wortmannin) was used to block PI3K signaling and its effects on CCI rats were tested. PI3K/Akt signaling increased in the spinal cord L4–L6 sections in the CCI rats. CCI also facilitated miniature excitatory postsynaptic potential of dorsal horn substantia gelatinosa neurons, increased phosphorylation of glutamate receptor subunit GluA1 and synapsin at the synapse, and induced mechanic allodynia. Wortmannin reversed biochemical, electrical, and behavioral changes in CCI rats. This study is the first to show PI3K/Akt signaling is required for spinal central sensitization in the CCI neuropathic pain model.     

New records of nematomorph parasites (Nematomorpha: Gordiida) of ground beetles (Coleoptera: Carabidae) and camel crickets (Orthoptera: Rhaphidophoridae) in Washington State

From 1998 to 2003, beetles and crickets infected with hairworms were collected from 4 localities within the Hanford Nuclear Site and the Hanford Reach National Monument, located in a shrub-steppe region of Washington State along the Columbia River. Infected hosts comprised 6 species of carabid beetles within 5 genera and 2 camel crickets within 1 genus; all are newly documented insect-nematomorph associations. A large proportion of the infected hosts (48%) were collected from a single site during a single collecting period. Of the 38 infected hosts, 32 contained a single worm, 4 hosts contained 2 worms, and 2 hosts contained 3 worms. Five of the hosts with multiple infections contained at least 1 male and 1 female worm. Camel crickets were infected with Neochordodes occidentalis while carabids were infected with an undescribed species of Gordionus . As the majority of hairworms are collected in the post-parasitic adult phase, host data and hairworm-arthropod associations remain poorly documented and our work adds new data to this area of nematomorph biology.     

Mechanical Conflict System: A Novel Operant Method for the Assessment of Nociceptive Behavior

A new operant test for preclinical pain research, termed the Mechanical Conflict System (MCS), is presented. Rats were given a choice either to remain in a brightly lit compartment or to escape to a dark compartment by crossing an array of height-adjustable nociceptive probes. Latency to escape the light compartment was evaluated with varying probe heights (0, .5, 1, 2, 3, and 4 mm above compartment floor) in rats with neuropathic pain induced by constriction nerve injury (CCI) and in naive control rats. Escape responses in CCI rats were assessed following intraperitoneal administration of pregabalin (10 and 30 mg/kg), morphine (2.5 and 5 mg/kg), and the tachykinin NK1 receptor antagonist, RP 67580 (1 and 10 mg/kg). Results indicate that escape latency increased as a function of probe height in both naive and CCI rats. Pregabalin (10 and 30 mg/kg) and morphine (5 mg/kg), but not RP 67580, decreased latency to escape in CCI rats suggesting an antinociceptive effect. In contrast, morphine (10 mg/kg) but not pregabalin (30 mg/kg) increased escape latency in naive rats suggesting a possible anxiolytic action of morphine in response to light-induced fear. No order effects following multiple test sessions were observed. We conclude that the MCS is a valid method to assess behavioral signs of affective pain in rodents.     

Comparison of the antinociceptive effects of ibuprofen arginate and ibuprofen in rat models of inflammatory and neuropathic pain

AimsIbuprofen arginate is a highly soluble salt formed by combining racemic ibuprofen with the amino acid l-arginine. This formulation is absorbed faster, and it is safe and effective in treating many forms of mild to moderate pain. We compared the analgesic effect of ibuprofen arginate and conventional ibuprofen in rat models of pain.Main methodsMechanical and cold allodynia were assessed in the chronic constriction injury (CCI) model of neuropathic pain, and mechanical allodynia was also examined in capsaicin-injected rats (a model of central sensitization). Inflammatory hypersensitivity was assessed with the formalin test. Ibuprofen-l-arginine, ibuprofen, l-arginine or saline was administered orally on a daily basis after CCI or capsaicin injection, and the von Frey and cold plate tests were performed on days 1, 3 and 7 after CCI or capsaicin administration. In the formalin-induced inflammatory pain test, the drugs were administered 30 min before formalin injection.Key findingsIbuprofen only exerts an antinociceptive effect in the formalin model whereas ibuprofen-l-arginine exerts antinociceptive effects on both mechanical and cold allodynia induced by CCI, mechanical allodynia induced by capsaicin injection, and in phase 2 of the formalin test, exhibiting superior antinociceptive activity to ibuprofen in all these tests. l-Arginine only exerted antinociceptive effects on cold allodynia in CCI.SignificanceThese results demonstrate that ibuprofen arginate has stronger antinociceptive effects than ibuprofen in all the models used, suggesting it might improve the therapeutic management of neuropathic and inflammatory pain.     

Ground beetles (Coleoptera,Carabidae) of the Hanford Nuclear Site in south-central Washington State

In this paper we report on ground beetles (Coleoptera: Carabidae) collected from the Hanford Nuclear Reservation and Hanford National Monument (together the Hanford Site), which is located in south-central Washington State. The Site is a relatively undisturbed relict of the shrub-steppe habitat present throughout much of the western Columbia Basin before the westward expansion of the United States. Species, localities, months of capture, and capture method are reported for field work conducted between 1994 and 2002. Most species were collected using pitfall traps, although other capture methods were employed. Trapping results indicate the Hanford Site supports a diverse ground beetle community, with over 90% of the 92 species captured native to North America. Four species collected during the study period are newly recorded for Washington State: Bembidion diligens Casey, Calosoma obsoletum Say, Pseudaptinus rufulus (LeConte), and Stenolophus lineola (Fabricius). Based on these data, the Site maintains a diverse ground beetle fauna and, due to its size and diversity of habitats, is an important repository of shrub-steppe biodiversity.     

A basic bifurcation structure from bursting to spiking of injured nerve fibers in a two-dimensional parameter space

Two different bifurcation scenarios of firing patterns with decreasing extracellular calcium concentrations were observed in identical sciatic nerve fibers of a chronic constriction injury (CCI) model when the extracellular 4-aminopyridine concentrations were fixed at two different levels. Both processes proceeded from period-1 bursting to period-1 spiking via complex or simple processes. Multiple typical experimental examples manifested dynamics closely matching those simulated in a recently proposed 4-dimensional model to describe the nonlinear dynamics of the CCI model, which included most cases of the bifurcation scenarios. As the extracellular 4-aminopyridine concentrations is increased, the structure of the bifurcation scenario becomes more complex. The results provide a basic framework for identifying the relationships between different neural firing patterns and different bifurcation scenarios and for revealing the complex nonlinear dynamics of neural firing patterns. The potential roles of the basic bifurcation structures in identifying the information process mechanism are discussed.     

Aerobic biodegradation of 4-methylquinoline by a soil bacterium.

Methylquinolines and related N-heterocyclic aromatic compounds are common contaminants associated with the use of hydrocarbons in both coal gasification and wood treatment processes. These compounds have been found in groundwater, and many are known mutagens. A stable, five-member bacterial consortium able to degrade 4-methylquinoline was established by selective enrichment using soil collected from an abandoned coal gasification site. The consortium was maintained for 5 years by serial transfer in a medium containing 4-methylquinoline. A gram-negative soil bacterium, strain Lep1, was isolated from the consortium and shown to utilize 4-methylquinoline as a source of carbon and energy during growth in liquid medium. A time course experiment demonstrated that both the isolate Lep1 and the consortium containing Lep1 were able to degrade 4-methylquinoline under aerobic conditions. Complete degradation of 4-methylquinoline by either strain Lep1 alone or the consortium was characterized by the production and eventual disappearance of 2-hydroxy-4-methylquinoline, followed by the appearance and persistence of a second metabolite tentatively identified as a hydroxy-4-methylcoumarin. Currently, there is no indication that 4-methylquinoline degradation proceeds differently in the consortium culture compared with Lep1 alone. This is the first report of 4-methylquinoline biodegradation under aerobic conditions.     

Effect of 1‐methyl‐D‐tryptophan and adoptive transfer of dendritic cells on polymicrobial sepsis induced by cecal content injection

A mouse model of polymicrobial sepsis induced by cecal content injection (CCI) was developed with the aim of gaining a better understanding of the mechanism of sepsis. This model has a similar survival pattern to the conventional model with the added benefits of ability to vary the severity of sepsis and greater consistency. Administration of 1‐methyl‐D ‐tryptophan (1‐MT) to inhibit indoleamine 2,3‐dioxygenase (IDO) in mice with CCI‐induced sepsis increased the survival rate and tended to up‐regulate IL‐10/IL‐12 serum concentrations. The effectiveness of 1‐MT was confirmed by increases in IL‐10 over IL‐12 in bone marrow‐derived dendritic cells (BMDCs) treated with LPS and 1‐MT and a superior survival rate 24 hr after injection of these double treated BMDCs in the CCI‐induced sepsis model. Therefore, CCI is both a useful and reliable technique for investigating polymicrobial sepsis. The present findings using this newly developed model suggest that inhibition of IDO alleviates the severity of polymicrobial sepsis and modulates the immune response even in cases of severe systemic septic inflammation.     

Isolation, characterization, and nitrification potential of a methylotroph and two heterotrophic bacteria from a consortium showing methane-dependent nitrification

Abstract A methanotrophic nitrifying consortium was previously obtained from a humisol which showed CH₄-dependent nitrification. Although the methanotroph could not be obtained in pure culture, three other members of the consortium have been isolated: An obligately methylotrophic Methylobacillus (Is-1) which grows only on CH₃OH and does not nitrify; a Pseudomonas (Is-2) which grows on Is-1 culture filtrate and produces NO₂⁻, NO₃⁻ and N₂O from NH₂OH, and NO₃⁻ from NO₂⁻; and a second Pseudomonas (Is-3) which produces NO₃⁻ from NH₄⁺ or NO₂⁻, and N₂O from NH₂OH. A model is proposed for the trophic relations and nitrogen transformations in the consortium which may apply to some natural systems.     

Decreased Expression and Role of GRK6 in Spinal Cord of Rats After Chronic Constriction Injury

Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.     

The role and mechanism of the up-regulation of fibrinolytic activity in painful peripheral nerve injury

Peripheral nerve injury is followed by Wallerians degeneration (WD) and degradation and regeneration of the extracellular matrix (ECM) are very important events in these processes. We tested fibrinolytic activities and the expression level of tPA and uPA levels in chronic constriction injury (CCI) model. The presented data demonstrates that in the injury nerve of CCI model, the fibrinolytic activities is upregulated and main caused by the upregulation of tPA expression. We also find that the activities of tPA and MMP-9 are co-upregulated post-CCI, both at CCI site and proximal site. These results indicate that tPA activity may regulate the MMP-9 activity in injury nerve post-CCI.     

Could CCI or FBCI Fully Eliminate the Impact of Curve Flexibility When Evaluating the Surgery Outcome for Thoracic Curve Idiopathic Scoliosis Patient? A Retrospective Study

PurposeTo clarify if CCI or FBCI could fully eliminate the influence of curve flexibility on the coronal correction rate.MethodsWe reviewed medical record of all thoracic curve AIS cases undergoing posterior spinal fusion with all pedicle screw systems from June 2011 to July 2013. Radiographical data was collected and calculated. Student t test, Pearson correlation analysis and linear regression analysis were used to analyze the data.Results60 were included in this study. The mean age was 14.7y (10-18y) with 10 males (17%) and 50 females (83%). The average Risser sign was 2.7. The mean thoracic Cobb angle before operation was 51.9°. The mean bending Cobb angle was 27.6° and the mean fulcrum bending Cobb angle was 17.4°. The mean Cobb angle at 2 week after surgery was 16.3°. The Pearson correlation coefficient r between CCI and BFR was -0.856(P<0.001), and between FBCI and FFR was -0.728 (P<0.001). A modified FBCI (M-FBCI) = (CR-0.513)/BFR or a modified CCI (M-CCI) = (CR-0.279)/FFR was generated by curve estimation has no significant correlation with FFR (r=-0.08, p=0.950) or with BFR (r=0.123, p=0.349).ConclusionsFulcrum-bending radiographs may better predict the outcome of AIS coronal correction than bending radiographs in thoracic curveAIS patients. Neither CCI nor FBCI can fully eliminate the impact of curve flexibility on the outcome of correction. A modified CCI or FBCI can better evaluating the corrective effects of different surgical techniques or instruments.     

Diversity and partitioning of bacterial populations within the accessory nidamental gland of the squid Euprymna scolopes

Microbial consortia confer important benefits to animal and plant hosts, and model associations are necessary to examine these types of host/microbe interactions. The accessory nidamental gland (ANG) is a female reproductive organ found among cephalopod mollusks that contains a consortium of bacteria, the exact function of which is unknown. To begin to understand the role of this organ, the bacterial consortium was characterized in the Hawaiian bobtail squid, Euprymna scolopes, a well-studied model organism for symbiosis research. Transmission electron microscopy (TEM) analysis of the ANG revealed dense bacterial assemblages of rod- and coccus-shaped cells segregated by morphology into separate, epithelium-lined tubules. The host epithelium was morphologically heterogeneous, containing ciliated and nonciliated cells with various brush border thicknesses. Hemocytes of the host's innate immune system were also found in close proximity to the bacteria within the tubules. A census of 16S rRNA genes suggested that Rhodobacterales, Rhizobiales, and Verrucomicrobia bacteria were prevalent, with members of the genus Phaeobacter dominating the consortium. Analysis of 454-shotgun sequencing data confirmed the presence of members of these taxa and revealed members of a fourth, Flavobacteria of the Bacteroidetes phylum. 16S rRNA fluorescent in situ hybridization (FISH) revealed that many ANG tubules were dominated by members of specific taxa, namely, Rhodobacterales, Verrucomicrobia, or Cytophaga-Flavobacteria-Bacteroidetes, suggesting symbiont partitioning to specific host tubules. In addition, FISH revealed that bacteria, including Phaeobacter species from the ANG, are likely deposited into the jelly coat of freshly laid eggs. This report establishes the ANG of the invertebrate E. scolopes as a model to examine interactions between a bacterial consortium and its host.     

A single dose of liposome-encapsulated hydromorphone provides extended analgesia in a rat model of neuropathic pain

Opioids have been shown to relieve thermal hyperalgesia associated with neuropathic pain. We used a novel technique to produce liposome-encapsulated hydromorphone (LEH), which we then tested in a chronic constriction injury (CCI) thermal hyperalgesia model of neuropathic pain. Rats were divided into sham-operated and CCI groups. Treatments consisted of LEH or standard hydromorphone, administered at surgery or 3 d after surgery, when thermal hyperalgesia had developed in the CCI rats. We measured thermal withdrawal latencies on days 0, 3, and 5. CCI rats given liposome-encapsulated vehicle or standard hydromorphone at surgery developed full thermal hyperalgesia. CCI rats given LEH at surgery exhibited no significant change compared with baseline values in thermal withdrawal latency, indicating that this preparation prevented hyperalgesia after a single injection. CCI rats given LEH on day 3 (that is, after they had developed hyperalgesia) showed reversal of hyperalgesia that persisted to day 5, whereas CCI rats given standard hydromorphone on day 3 showed only brief (approximately 90 min) reversal of hyperalgesia. Preemptive injection of LEH prevented hyperalgesia in this model for as long as 5 d. In addition, hyperalgesia was alleviated for at least 2 d after injection of a single dose of LEH. These results suggest that liposome-encapsulation of hydromorphone offers a convenient and effective means to provide relief from neuropathic pain in this rodent model.     

Metabolism of the 18O-methoxy substituent of 3-methoxybenzoic acid and other unlabeled methoxybenzoic acids by anaerobic bacteria.

O-methyl substituents of aromatic compounds can provide C1 growth substrates for facultative and strict anaerobic bacteria isolated from diverse environments. The mechanism of the bioconversion of methoxylated benzoic acids to the hydroxylated derivatives was investigated with a model substrate and cultures of one anaerobic consortium, eight strict anaerobic bacteria, and one facultative anaerobic microorganism. Using high-pressure liquid chromatography and gas chromatography-mass spectral analysis, we found that a haloaromatic dehalogenating consortium, a dehalogenating isolate from that consortium, Eubacterium limosum, and a strain of Acetobacterium woodii metabolized 3-[methoxy-18O]methoxybenzoic acid (3-anisic acid) to 3-[hydroxy-18O]hydroxybenzoic acid stoichiometrically at rates of 1.5, 3.2, 52.4, and 36.7 nmol/min per mg of protein, respectively. A different strain of Acetobacterium and strains of Syntrophococcus, Clostridium, Desulfotomaculum, Enterobacter, and an anaerobic bacterium, strain TH-001, were unable to transform this compound. The O-demethylating ability of E. limosum was induced only with appropriate methoxylated benzoates but not with D-glucose, lactate, isoleucine, or methanol. Cross-acclimation and growth experiments with E. limosum showed a rate of metabolism that was an order of magnitude slower and showed no growth with either 4-methoxysalicylic acid (2-hydroxy-4-methoxybenzoic acid) or 4-anisic acid (4-methoxybenzoic acid) when adapted to 3-anisic acid. However, A. woodii NZva-16 showed slower rates and no growth with 3- or 4-methoxysalicylic acid when adapted to 3-anisic acid in similar experiments. The results clearly indicate a methyl rather than methoxy group removal mechanism for such reactions.     

The Effect and Mechanism of Growth Hormone Replacement on Cognitive Function in Rats with Traumatic Brain Injury

Objective

The effects of growth hormone on cognitive dysfunction were observed in a controlled cortical impact (CCI) rat model and the underlying mechanism was explored.

Method

Three-month-old male SD rats were randomly divided into sham (n = 10), control (n = 10), and CCI groups (n = 40) The parameters were set as follows: striking speed, 3.5 m/s; impact depth, 1.5 mm; and dwell time, 400 msec. Eight and ten weeks post-injury, the GH levels were measured the water maze test and novel object recognition test were performed. CCI rats were divided into normal and decreased GH groups, and further randomly divided into two sub-groups (rhGH treatment and saline vehicle groups). All rats were tested for SYN, BDNF, and TrkB mRNA in the prefrontal cortex and hippocampus by RT-PCR.

Results

CCI rats 8 weeks post-injury had cognitive dysfunction regardless of the GH level (P<0.05). rhGH treatment improved cognitive function in CCI rats. There was a positive correlation between the expression of prefrontal BDNF and SYN mRNA in CCI rats after rhGH therapy and the water maze test score (r = 0.773 and 0.534, respectively; P<0.05). Furthermore, the expression of BDNF, TrkB, and SYN mRNA in the hippocampus was negatively correlated with the water maze test score (r = 0.602, 0.773, 0.672, and 0.783, respectively; P<0.05). There was a difference in the expression of hippocampal and prefrontal BDNF, TrkB, and SYN mRNA (P<0.05)

Conclusion

rhGH treatment had a positive effect on cognitive function, which was more evident in GH-deficient rats. The increased expression of hippocampal and prefrontal BDNF and TrkB mRNA is implicated in rhGH therapy to improve cognitive function. Changes in the expression of hippocampal SYN mRNA following rhGH therapy may also play a role in improving cognitive function.