Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

Some factors influencing the neurotoxicity of intrastriatal injections of kainic acid

Intrastriatal injections of kainic acid are known to destroy striatal neurons including many containing choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Using these enzymes as indices of neuronal loss, the neurotoxicity of small doses of kainic acid was found to be influenced by injection time and volume. It was partly blocked by coninjection of some but not all glutamate antagonists or by prior lesioning of the corticostriatal tract. Other adjuvants, drugs, or lesions tested had little modifying effect, except that changes in the dopaminergic system seemed to increase the toxicity towards cholinergic but not GABAnergic systems. High-affinity glutamate accumulation by neostriatal synaptosomes was significantly increased 1–7 days following kainic acid injections. MAO and acetylcholinesterase activities were depressed in kainic acid-lesioned striata but not nearly as much as were CAT and GAD. An indirect mechanism involving glutamate release and inhibition of reuptake is suggested for kainic acid neurotoxicity.     

粒细胞-巨噬细胞集落刺激因子微球制剂治疗肿瘤的研究

目的：评价粒细胞－巨噬细胞集落刺激因子（GM—csF）微球剂型在肿瘤动物模型治疗作用。方法：建立肿瘤动物模型,微球剂型对该肿瘤模型治疗效果考察及毒性评价,同时与市售的注射水溶液剂型进行比较。结果：微球剂型和市售水溶液注射剂型同剂量对肿瘤模型治疗效果,微球剂型的效果明显好于市售的;同时微球剂型的毒性也小于市售的。结论：粒细胞－巨噬细胞集落刺激因子微球能提高GM—CSF治疗效果,同时降低毒性。     

Neuroprotective Actions of the Synthetic Estrogen 17α-Ethynylestradiol in the Hippocampus

17α-Ethynylestradiol (EE2), a major constituent of many oral contraceptives, is similar in structure to 17β-estradiol, which has neuroprotective properties in several animal models. This study explored the potential neuroprotective actions of EE2 against kainic and quinolinic acid toxicity in the hippocampus of adult ovariectomized Wistar rats. A decrease in the number of Nissl-stained neurons and the induction of vimentin immunoreactivity in astrocytes was observed in the hilus of the dentate gyrus of the hippocampus after the administration of either kainic acid or quinolinic acid. EE2 prevented the neuronal loss and the induction of vimentin immunoreactivity induced by kainic acid at low (1 μg/rat) and high (10–100 μg/rat) doses and exerted a protection against quinolinic acid toxicity at a low dose (1 μg/rat) only. These observations demonstrate that EE2 exerts neuroprotective actions against excitotoxic insults. This finding is relevant for the design of new neuroprotective estrogenic compounds.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

Comparison of anticonvulsant effect of ethanol against NMDA-, kainic acid- and picrotoxin-induced convulsions in rats

The anticonvulsant effect of ethanol against N-methyl-D-aspartic acid-(NMDA), kainic acid-, and picrotoxin-induced convulsions was studied in rats. Ethanol (2 g/kg, ip) offered protection against these agents, and it was most effective against picrotoxin and least effective against kainic acid. MK801, NMDA receptor antagonist, also provided protection against these agents. However, it was most effective against NMDA and least effective against kainic acid. Ineffective doses of MK801 (0.1 mg/kg, ip) and ethanol (0.5 g/kg, ip), when administered concurrently, had a facilitatory anticonvulsant effect, thereby providing protection against mortality following severe convulsions induced by NMDA or picrotoxin, but not against kainic acid. The protective effect of ethanol against NMDA- and kainic acid-induced neurotoxicity, in contrast to picrotoxin-induced toxicity, was not reversed by imidazodiazepine, Ro 15-4513, an ethanol antagonist. Furthermore, Ro 15-4513 did not produce any proconvulsant effect with NMDA or kainic acid. These findings suggested that the anticonvulsant actions of ethanol may be attributed to its ability to antagonize NMDA-mediated excitatory responses and facilitate the GABAergic transmission.     

Effect of intrahippocampal kainic acid on the behavior of rats and functional state of mitochondria in brain structures

Cognitive processes and functional state of mitochondria in brain structures of Wistar rats were studied after intrahippocampal injection of kainic acid, an agonist of glutamate receptors. A single administration of 0.25 μg kainic acid into the dorsal part of the left and right hippocampi affected task retrieval and decreased inhibition of unrewarded responses. The injection of 0.75 μg kainic acid induced recurrent seizures and completely disorganized animal behavior. The functional state of mitochondria, as an important marker of excitotoxicity, was studied after intrahippocampal injections of kainic acid in the same doses. Kainic acid at 0.25 μg proved to activate the oxidative phosphorylation in hippocampal mitochondria. A higher (epileptogenic) dose of kainic acid inhibited mitochondrial respiration in the frontal cortex, but had an insignificant effect on mitochondrial respiration in the hippocampus. The disturbed interaction between the hippocampal system and frontal cortex after kainic acid administration can be the main factor of the revealed cognitive dysfunctions.     

Choline Acetyltransferase Activity in the Rat Brain Cortex Homogenate, Synaptosomes, and Capillaries After Lesioning the Nucleus Basalis Magnocellularis

Stereotaxic lesions of the nucleus basalis magnocellularis were made unilaterally in male Wistar rats with either kainic or ibotenic acid, using the contralateral side as control. Differences in behavior, body weight, and survival were observed between the kainic and ibotenic acid-treated rats. One week after surgery, the rats were sacrificed and the effect of the lesions on choline acetyltransferase activity was measured in brain cortex homogenate, synaptosomes, and capillaries. In kainic acid-lesioned rats, choline acetyltransferase activity decreased in homogenate and synaptosomes of the ipsilateral side with respect to that of the contralateral side; but the ibotenic acid lesion, which also reduced the ipsilateral choline acetyltransferase activity in homogenate, showed a rather different effect on the enzymatic activity of the synaptosomes. There were also differences between the effect of kainic and ibotenic acid lesions on choline acetyltransferase activity in the capillaries of the ipsilateral side with respect to that of the contralateral one. However, capillary choline acetyltransferase activity of the treated rats was in both sides three times higher than that of unoperated rats.     

Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus

The effect of local administration of kainic acid in the rabbit hippocampus was studied; the hippocampus was perfused continuously in the freely moving animal with an implanted 0.3-mm dialysis fiber. The pattern of endogenous amino acids in the perfusate, reflecting extracellular amino acids, was monitored with liquid chromatography separation and fluorimetric detection of amino acid derivatives. Kainic acid was included in the perfusion medium for up to 70 min at 0.1-1.0 mM and, with time, induced epileptiform activity. Endogenous glutamic acid, taurine, and phosphoethanolamine levels were increased selectively at the lower perfusion concentrations of kainic acid. Long perfusion periods with higher concentrations increased the levels of virtually all amino acids. Perfusion of the hippocampus with depolarizing concentrations of potassium gave an amino acid response partly similar to that seen with kainic acid treatment. However, one notable difference between the two responses was that the extracellular concentration of glutamine, although not influenced by kainic acid, was significantly decreased after high potassium concentrations. These results confirm previous notions that kainic acid has a primarily excitatory effect, one manifestation of this effect being the release of glutamic acid.     

Corticosterone Enhances Kainic Acid-Induced Calcium Elevation in Cultured Hippocampal Neurons

Abstract: Corticosterone, a steroid secreted during stress, increases hippocampal neuronal vulnerability to excitotoxins, hypoxia-ischemia, and antimetabolites. Energy supplementation and N -methyl-d-aspartate receptor antagonists prevent this corticosterone-enhanced neurotoxicity. Because neuronal calcium regulation is energy dependent and a large calcium influx accompanies N -methyl-d-aspartate receptor activation, we investigated whether corticosterone exacerbates the elevation of hippocampal neuronal calcium induced by the glutamatergic excitotoxin kainic acid. Corticosterone caused a 23-fold increase in the magnitude of the calcium response to kainic acid, a sevenfold increase in the peak magnitude of the calcium response, and a twofold increase in calcium recovery time. This corticosterone effect may be energetic in nature as corticosterone decreases hippocampal neuronal glucose transport. Glucose supplementation reduced the corticosterone effect on the magnitude and peak magnitude of the calcium response to kainic acid. Glucose reduction, by the approximate magnitude by which corticosterone inhibits glucose transport, mimicked the corticosterone effect on the peak magnitude of the calcium response to kainic acid. Thus, corticosterone increases calcium after kainic acid exposure in hippocampal neurons in an energy-dependent manner. Elevated calcium is strongly implicated in stimulating neurotoxic cascades during other energetic insults and may be the mechanism for the corticosterone-induced hippocampal neuronal vulnerability and toxicity.     

Characterization of specific in vivo binding of neuroleptic drugs in rat brain.

Lesioning of the rat striatum with kainic acid may provide a useful animal model with which to study Huntington's Disease since, in both situations, changes in several neurochemical parameters appear similar. In this study, we examined the time course of dopaminergic (DA) and muscarinic cholinergic (MCHOL) receptor alterations after kainic acid injection into the rat striatum. As early as two days after unilateral, intrastriatal injection of kainic acid, most striatal perikaya in the injected area had been destroyed as seen by histological examination. A progressive decrease in the DA and MCHOL receptors continued which was not due to changes in their affinity for their respective receptors. By 48 days after injection, there was about 75% decrease in DA receptors and about a 65% decrease in MCHOL receptors. The DA receptor loss is similar in extent to the reported loss in activity of striatal, dopamine-stimulated adenylate cyclase after kainic acid lesion. The DA and MCHOLreceptor loss is similar to the reported loss of neostriatal DA and MCHOL receptors in Huntington's Disease.     

Muslims in the 2001 Census of England and Wales: Gender and economic disadvantage

The article presents new data for the Muslim population of Britain from the 2001 Census. It uses the cross tabulations of ethnicity by religion to back-project the growth of the Muslim population from 21,000 in 1951 to 1.6 millions in 2001. It examines the social, economic, demographic and geographic characteristics of the population. Although Muslims are often represented as a homogenous group, there are considerable internal differences, so that the characteristics of the population as a whole do not apply to all groups within. The 2001 Census shows that two-thirds of British Muslims are ethnically Pakistani, Indian and Bangladeshi, but one-third comes from diverse European, African, North African, Middle Eastern and other Asian sources. Nevertheless, Muslim gender roles emerge as a critical differentiator of socio-economic vulnerability. Taken as a whole, the Muslim population is young and rapidly growing; its socio-economic profile is depressed, marked by the exceptionally low participation rate of women in the formal labour market, and by high concentration in areas of multiple deprivation.     

Neuropharmacological profile of FrPbAII, purified from the venom of the social spider Parawixia bistriata (Araneae, Araneidae), in Wistar rats

The aims of the present study were to investigate the anticonvulsant activity and behavioral toxicity of FrPbAII using freely moving Wistar rats. Moreover, the effectiveness of this compound against chemical convulsants was compared to that of the inhibitor of the GABAergic uptake, nipecotic acid. Our results show that FrPbAII was effective against seizures induced by the i.c.v. injection of pilocarpine (ED(50) = 0.05 microg/animal), picrotoxin (ED(50) = 0.02 microg/animal), kainic acid (ED(50) = 0.2 microg/animal) and the systemic administration of PTZ (ED(50) = 0.03 microg/animal). The anticonvulsant effect of FrPbAII differed from that of nipecotic acid in potency, as the doses needed to block the seizures were more than 10 folds lower. Toxicity assays revealed that in the rotarod, the toxic dose of the FrPbAII is 1.33 microg/animal, and the therapeutic indexes were calculated for each convulsant. Furthermore, the spontaneous locomotor activity of treated animals was not altered when compared to control animals but differed from the animals treated with nipecotic acid. Still, FrPbAII did not induce changes in any of the behavioral parameters analyzed. Finally, when tested for cognitive impairments in the Morris water maze, the i.c.v. injection of FrPbAII did not alter escape latencies of treated animals. These findings indicate that the novel GABA uptake inhibitor is a potent anticonvulsant with mild side-effects when administered to Wistar rats.     

Incorporating concentration dependence in stable isotope mixing models

Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source linear mixing model assumes that the proportional contribution of a source to a mixture is the same for both elements (e.g., C, N). This may be a reasonable assumption if the concentrations are similar among all sources. However, one source is often particularly rich or poor in one element (e.g., N), which logically leads to a proportionate increase or decrease in the contribution of that source to the mixture for that element relative to the other element (e.g., C). We have developed a concentration-weighted linear mixing model, which assumes that for each element, a source's contribution is proportional to the contributed mass times the elemental concentration in that source. The model is outlined for two elements and three sources, but can be generalized to n elements and n+1 sources. Sensitivity analyses for C and N in three sources indicated that varying the N concentration of just one source had large and differing effects on the estimated source contributions of mass, C, and N. The same was true for a case study of bears feeding on salmon, moose, and N-poor plants. In this example, the estimated biomass contribution of salmon from the concentration-weighted model was markedly less than the standard model estimate. Application of the model to a captive feeding study of captive mink fed on salmon, lean beef, and C-rich, N-poor beef fat reproduced very closely the known dietary proportions, whereas the standard model failed to yield a set of positive source proportions. Use of this concentration-weighted model is recommended whenever the elemental concentrations vary substantially among the sources, which may occur in a variety of ecological and geochemical applications of stable isotope analysis. Possible examples besides dietary and food web studies include stable isotope analysis of water sources in soils, plants, or water bodies; geological sources for soils or marine systems; decomposition and soil organic matter dynamics, and tracing animal migration patterns. A spreadsheet for performing the calculations for this model is available at http://www.epa.gov/wed/pages/models.htm.     

A kainic acid receptor from frog brain purified using domoic acid affinity chromatography

A kainic acid receptor was purified from Triton X-100/digitonin-solubilized frog brain membranes. The purification was carried out in two steps: ion exchange chromatography using DEAE-Sepharose CL-6B and affinity chromatography with domoic acid immobilized on Sepharose 4B. The specific binding activity of the affinity-purified receptor is 481-fold higher than that of the crude solubilized preparation and 1617-fold higher than that of the whole membrane fraction. Scatchard analyses of the affinity-purified receptor showed a curvilinear plot which fit a two-site model with dissociation constants of 5.5 and 34 nM and Bmax values of 1700 pmol/mg protein and 4400 pmol/mg protein for the high and low affinity components, respectively. The dissociation constants of the purified receptor are similar to those of the crude soluble preparation (4.8 and 39 nM). Inhibition constants for several kainic acid analogs were also similar for the purified and crude preparations. The active purified receptor migrated with a Mr = 570,000 on gel filtration analysis using Sepharose 6B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor showed a single broad band with silver stain, migrating with a Mr = 48,000.     

芽孢杆菌(Bacillus sp.)脂肽抗生素发酵工艺及分离纯化

微生物脂肽具有抗菌谱广、热稳定性高、低毒、低抗药性等优点,近年来受到国内外广泛关注。综述了芽孢杆菌脂肽抗生素的发酵和分离纯化工艺的最新研究进展。在发酵工艺中,培养基营养组成、发酵温度、搅拌转速和通气量等参数对脂肽的产量至关重要,碳源、氮源和金属离子的组成与配比都会影响芽孢杆菌的生长与产物的合成,适当控制搅拌转速和通气量可提高脂肽产量。此外,近年来一些新型发酵工艺,如泡沫回流、固定化细胞、无泡发酵、固态发酵等被用于脂肽生产,通过改进发酵方式,在降低成本的同时提高了脂肽抗生素产量。抗菌脂肽分离纯化的主要方法包括超滤、吸附、泡沫分离及色谱法等,这些方法相对于传统的酸沉和萃取,具有可连续生产、脂肽提取量高及成本低等优点。同时,多种纯化方法的组合应用大幅度提高了抗菌脂肽的提取效果,有效降低了成本,是脂肽抗生素分离提取的发展方向。     

Study of Differential Effects of Kainic Acid on Metabolic Rates, Utilizing Exogenous or Endogenous Substrates, in Rat Brain Slices

CO2 production from exogenous glucose of cortical, whole hippocampal, and CA3 region hippocampal slices, as well as O2 consumption of whole hippocampal slices, were measured in the presence of different concentrations of kainic acid. A moderate, significant increase of CO2 production was seen only in the CA3 region hippocampal preparation at kainic acid concentrations of 10(-4)-10(-2) M. The O2 consumption, at the expense of endogenous energy stores of whole hippocampal slices, was substantially increased by 10(-3) M kainic acid when the slices were incubated without exogenous glucose. The effect was partly paralleled by the use of high (50 mM) K+ concentration. Some of the possible factors involved in the differential metabolic responses of brain slices to the action of kainic acid are discussed briefly.     

Source tracking of Escherichia coli by 16S-23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of the rrnB ribosomal operon

This research validates a novel approach for source tracking based on denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from Escherichia coli isolates. Escherichia coli from different animal sources and from river samples upstream from, at, and downstream of a combined sewer overflow were subjected to DGGE to determine sequence variations within the 16S-23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operon. To prevent amplification of all 7 E. coli ribosomal operons by PCR amplification using universal primers, sequence-specific primers were utilized for the rrnB operon. Another primer set was then used to prepare samples of the 16S-23S ISR for DGGE. Comparison of PCR-DGGE results between human and animal sources revealed differences in the distribution and frequency of the DGGE bands produced. Human and Canada Goose isolates had the most unique distribution patterns and the highest percent of unique isolates and were grouped separately from all other animal sources. Method validation suggests that there are enough host specificity and genetic differences for use in the field. Field results at and around a combined sewer overflow indicate that this method can be used for microbial source tracking.