The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.     

Turnaround of Axoplasmic Transport of Selected Particle-Specific Enzymes at an Injury in Control and Diisopropylphosphorofluoridate-Treated Rats

: Reversal of direction (turnaround) of axonal transport of particle specific enzyme activities was studied at a ligature placed on rat sciatic nerve. In the principal experiment, the ligature remained on the nerve in vivo several hours, allowing enzyme activities (acetylcholinesterase, acid phosphatase, and monoamine oxidase) to accumulate immediately proximal to the tie. The nerve was then tied a second time, proximal to the first tie, and incubated in vitro for several more hours. Accumulation of enzyme activities just distal to the second tie was measured. This second accumulation, of activities traveling in the retrograde direction, was shown to be the result of turnaround in several ways. (1) The increase in activity distal to the second tie was equal to the decrease in activity proximal to the first. (2) The increase in enzyme activities distal to the second tie was greatly reduced when the accumulation proximal to the first tie was trapped by placing a third tie between the first and second ties. (3) It was shown that the activity that accumulated distal to the second tie could not have been in retrograde motion at the time of the first tie. (4) Accumulation distal to the second tie was not a function of the length of nerve segment included between the two ties. In contrast to the consistent occurrence of turnaround of orthograde flow, turnaround of retrograde flow could not be demonstrated. Turnaround transport was blocked by incubation in the cold and in the presence of NaCN or vinblastine. The turnaround process operated on all three enzymes studied, suggesting that it operates on lysosomes and mitochondria, as well as on the endoplasmic reticulum-like material bearing acetylcholinesterase. Evidence for the participation of the transport process in the renewal of AChE in the distal portions of the axon was obtained in experiments using diisopropylphosphorofluoridate and cycloheximide.     

Estrogenic and antiestrogenic activities of 16alpha- and 2-hydroxy metabolites of 17beta-estradiol in MCF-7 and T47D human breast cancer cells

The comparative mitogenic activities of 17beta-estradiol (E2) and four metabolites, 2-hydroxyestradiol (2-OHE2), 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestradiol (16alpha-OHE2) and 16alpha-hydroxyestrone (16alpha-OHE1) were determined in estrogen receptor (ER)-positive MCF-7 and T47D human breast cancer cells. E2 (1 nM) induced a 7- to 13-fold increase in cell number in both cell lines compared to untreated cells and the mitogenic potencies of 16alpha-OHE1 or 16alpha-OHE2 were comparable to or greater than E2. In contrast, 2-OHE1 and 2-OHE2 were weak mitogens in both cell lines and in cells cotreated with 1 nM E2 and 100 or 1000 nM 2-OHE1 or 2-OHE2, there was a significant inhibition of hormone-induced cell proliferation. The comparative ER agonist/antagonist activities of E2 and the metabolites on transactivation were determined in T47D cells transiently transfected with constructs containing promoter inserts from the cathepsin D (pCD) and creatine kinase B (pCKB) genes. E2, 16alpha-OHE2 and 16alpha-OHE1 induced reporter gene activity in both MCF-7 or T47D cells transfected with pCKB or pCD. In contrast, 2-OHE1 and 2-OHE2 did not exhibit ER agonist activity for these transactivation assays, but in cells cotreated with E2 plus 2-OHE1 or 2-OHE2, there was a significant decrease in the hormone-induced response. These results demonstrate that 16alpha-OHE1/16alpha-OHE2 exhibit estrogenic activities similar to that observed for E2, whereas the 2-catecholestrogens are weak ER agonists (cell proliferation) or antagonists (cell proliferation and transactivation).     

Greenhouse gas emissions and carbon sequestration potential in restored wetlands of the Canadian prairie pothole region

North American prairie pothole wetlands are known to be important carbon stores. As a result there is interest in using wetland restoration and conservation programs to mitigate the effects of increasing greenhouse gas concentration in the atmosphere. However, the same conditions which cause these systems to accumulate organic carbon also produce the conditions under which methanogenesis can occur. As a result prairie pothole wetlands are potential hotspots for methane emissions. We examined change in soil organic carbon density as well as emissions of methane and nitrous oxide in newly restored, long-term restored, and reference wetlands across the Canadian prairies to determine the net GHG mitigation potential associated with wetland restoration. Our results indicate that methane emissions from seasonal, semi-permanent, and permanent prairie pothole wetlands are quite high while nitrous oxide emissions from these sites are fairly low. Increases in soil organic carbon between newly restored and long-term restored wetlands supports the conclusion that restored wetlands sequester organic carbon. Assuming a sequestration duration of 33 years and a return to historical SOC densities we estimate a mean annual sequestration rate for restored wetlands of 2.7 Mg C ha⁻¹year⁻¹ or 9.9 Mg CO₂ eq. ha⁻¹ year⁻¹. Even after accounting for increased CH₄ emissions associated with restoration our research indicates that wetland restoration would sequester approximately 3.25 Mg CO₂ eq. ha⁻¹year⁻¹. This research indicates that widescale restoration of seasonal, semi-permanent, and permanent wetlands in the Canadian prairies could help mitigate GHG emissions in the near term until a more viable long-term solution to increasing atmospheric concentrations of GHGs can be found.     

Three-dimensional structure of the mini-M conotoxin mr3a

Conotoxin mr3a from the venom of Conus marmoreus, a novel peptide that induces rolling seizures in mice, has the peptide sequence GCCGSFACRFGCVOCCV, where O is trans-4-hydroxyproline, and the chain is cross-linked with disulfide bonds between Cys-2 and Cys-16, Cys-3 and Cys-12, and Cys-8 and Cys-15. The tertiary structure of mr3a was determined by 2D 1H NMR in combination with a standard distance-geometry algorithm. The final set of 22 structures for the peptide had a mean global backbone RMS deviation of 0.53 +/- 0.22 A based on 51 NOE, 6 hydrogen bond, 6 phi dihedral angle, and 3 disulfide bond constraints. Conotoxin mr3a is the first example of the new mini-M branch of conopeptides in the M superfamily. Members of the maxi-M branch, whose structures are known, include the mu- and psi-conotoxins, both of which share a common disulfide bond connectivity. Although mr3a has the same arrangement of Cys residues as the mu- and psi-conotoxins, its disulfide connectivity is different. This gives mr3a a distinctive "triple-turn" backbone.     

Controlling HIV Epidemics among Injection Drug Users: Eight Years of Cross-Border HIV Prevention Interventions in Vietnam and China

Introduction

HIV in Vietnam and Southern China is driven by injection drug use. We have implemented HIV prevention interventions for IDUs since 2002–2003 in Lang Son and Ha Giang Provinces, Vietnam and Ning Ming County (Guangxi), China.

Methods

Interventions provide peer education and needle/syringe distribution. Evaluation employed serial cross-sectional surveys of IDUs 26 waves from 2002 to 2011, including interviews and HIV testing. Outcomes were HIV risk behaviors, HIV prevalence and incidence. HIV incidence estimation used two methods: 1) among new injectors from prevalence data; and 2) a capture enzyme immunoassay (BED testing) on all HIV+ samples.

Results

We found significant declines in drug-related risk behaviors and sharp reductions in HIV prevalence among IDUs (Lang Son from 46% to 23% [p<0.001], Ning Ming: from 17% to 11% [p = 0.003], and Ha Giang: from 51% to 18% [p<0.001]), reductions not experienced in other provinces without such interventions. There were significant declines in HIV incidence to low levels among new injectors through 36–48 months, then some rebound, particularly in Ning Ming, but BED-based estimates revealed significant reductions in incidence through 96 months.

Discussion

This is one of the longest studies of HIV prevention among IDUs in Asia. The rebound in incidence among new injectors may reflect sexual transmission. BED-based estimates may overstate incidence (because of false-recent results in patients with long-term infection or on ARV treatment) but adjustment for false-recent results and survey responses on duration of infection generally confirm BED-based incidence trends. Combined trends from the two estimation methods show sharp declines in incidence to low levels. The significant downward trends in all primary outcome measures indicate that the Cross-Border interventions played an important role in bringing HIV epidemics among IDUs under control. The Cross-Border project offers a model of HIV prevention for IDUs that should be considered for large-scale replication.