Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.

The venoms from three spider species, Araneus gemma, Neoscona arabesca, and Argiope aurantia, were shown to inhibit the high-affinity, sodium-independent L-glutamate-binding sites in rat brain synaptic membranes. The same three venoms caused concentration-dependent inhibition of the activity of the glutamate-binding glycoprotein purified from rat brain synaptic membranes. The venom milked from the glands of Araneus gemma was the most active inhibitor of L-glutamate binding, causing 60-80% inhibition of both synaptic membrane and purified protein binding activity at 0.01 unit. The inhibitory activity of this venom was associated with a single protein peak obtained from gel permeation chromatography of the venom. Finally, the effect of the venom from Araneus gemma on the synaptic membrane glutamate-binding sites was slowly reversible. These observations indicate that the spider venoms have a direct effect on the recognition sites for L-glutamic acid in brain synaptic membranes and that these sites are related to the physiologic glutamate receptors.     

Dietary L-aspartate and L-glutamate inhibit fatty streak initiation in cholesterol-fed rabbit

BACKGROUND AND AIM: Low-density lipoprotein (LDL) oxidation is a potential atherogenic agent, and protecting LDL from oxidation prevents atherogenesis. It has been shown that L-aspartate and L-glutamate decrease lipid peroxidation after reoxygenation by means of the initiation of the cardiopulmonary bypass circuit (CPB), when supplemented to the CPB prime, and so they may protect against atherogenesis. The aim of this study was to evaluate the effect of the dietary administration of L-aspartate and L-glutamate on fatty streak onset in cholesterol-fed rabbit. METHODS AND RESULTS: Male New Zealand white rabbits were fed for four weeks with either a high-cholesterol plus corn oil diet (control group) or the same diet supplemented with 12.5 mM L-aspartate and 12.5 mM L-glutamate in drinking water (Asp + Glu group). The mononuclear cells adhering to the endothelium and the intimal foam cells of the thoracic aorta were used to quantify the extent of atherosclerosis. Total serum cholesterol and lipid peroxidation activity, measured as thiobarbituric acid reactive substances (TBARS), were determined 0, 1 and 4 weeks after a 2-week adaptation period. There were no between-group differences in body weight or food intake during the intervention. Serum TBARS were significantly increased in both groups during the experimental period but without any statistical difference between groups. At the end of the dietary intervention, there was a ten-fold increase in total serum cholesterol concentration in both groups vs baseline. The numbers of adherent mononuclear cells and intimal foam cells were both significantly lower in the Asp + Glu group. CONCLUSIONS: Our results suggest that dietary supplementation with L-aspartate and L-glutamate seems to protect the arterial wall from atherogenesis in an experimental animal.     

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.     

Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.

Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant.     

Differences in the luteinizing hormone and prolactin responses to multiple injections of kainate, as compared to N-methyl-D,L-aspartate, in cycling rats.

We have previously reported that repetitive iv injections of NMA [N-methyl-D,L-aspartate, the mixed analog acting on the N-methyl-D-aspartate (NMDA) receptor] can induce a consistent increase in LH and PRL secretion in cycling rats, but not in lactating rats. To further explore the use of excitatory amino acids (EAAs) as tools for understanding the regulation of the neuroendocrine reproductive axis, we have examined the effects of multiple injections of kainate, an agonist to another subclass of EAA receptor, on LH and PRL secretion in cycling rats. Recent studies suggest that kainate receptors may be more abundant than NMDA receptors in the hypothalamus. Five iv injections of kainate were administered at 50-min intervals to diestrous or estrous rats. Blood samples were collected every 10 min and assayed for LH and PRL. LH, but not PRL secretion, was stimulated by this regimen of kainate treatment. Surprisingly, the LH response to kainate, unlike NMA, decreased with repetitive injections of the drug. The response to the last pulse of kainate was approximately 30-40% of the first pulse. This decline in LH responsiveness to kainate was not due to desensitization at the level of the pituitary or to refractoriness of GnRH neurons, since further stimulation of LH release could be obtained by the administration of GnRH or NMA. The mechanisms responsible for the diminishing GnRH response to kainate remain unclear. However, we speculate that it might be due to the delayed activation of inhibitory inputs to GnRH neurons or to the desensitization of kainate receptors. On the other hand, the absence of a PRL response to kainate, in contrast to the stimulatory effect of NMA, most likely reflects differences in the distribution of kainate and NMDA receptors on dopamine neurons and neurons containing PRL-releasing factors, or on extrahypothalamic afferent neuronal populations projecting to the hypothalamus. In conclusion, the effects of systemic injections of kainate on LH and PRL secretion differed from NMA in that the LH response could not be sustained with multiple injections and PRL was unresponsive to kainate stimulation.     

(-)-baclofen and gamma-aminobutyric acid inhibit calcium currents in isolated retinal ganglion cells.

Of the various synaptic inputs known to converge upon retinal ganglion cells, the major inhibitory inputs are thought to be GABAergic. Although gamma-aminobutyric acid (GABA) is known to activate anion-selective ion channels in retinal ganglion cells, we have tested the possibility that GABA can also modulate cationic conductances in these cells, as seen in other central and peripheral neurons. Specifically, we have made whole-cell patch-clamp recordings to test whether voltage-gated calcium currents in isolated goldfish retinal ganglion cells are sensitive to GABAB receptor ligands. (-)-Baclofen and GABA inhibited calcium currents activated by moderately long depolarizations and, during large depolarizations (e.g., to 0 mV), also appeared to accelerate the rate of current decay. The calcium current inhibition induced by (-)-baclofen and GABA was not prevented by 2-hydroxysaclofen, phaclofen, or bicuculline, even though bicuculline suppressed a GABA-activated conductance in these cells. These results demonstrate the presence of baclofen- and GABA-sensitive calcium currents in vertebrate retinal ganglion cells as well as the coexistence of GABAA and GABAB receptors in individual retinal ganglion cells.     

Sequential exposures to ozone and lipopolysaccharide in postnatal lung enhance or inhibit cytokine responses

Sequential exposures to inhaled environmental pollutants may result in injuries/responses not predicted by evaluating exposures to an individual toxicant. This may indicate that the lung is damaged or primed by earlier events, so exposure to a nontoxic dose of an environmental pollutant may be sufficient to trigger adverse responses. The present study was designed to test the hypothesis that stimulating lung epithelial damage or inflammatory cell activation followed by a second stimulus leads to responses not seen after individual exposures in the postnatal lung. C57Bl/6 mice ages 4, 10, and 56 days were exposed to either a 10-minute inhalation of lipopolysaccharide (LPS), with an estimated deposited dose of 26 EU, followed immediately by 2.5 PPM ozone for 4 hours, or to 2.5 PPM ozone for 4 hours followed immediately by a 10-minute inhalation of LPS and examined 2 hours post exposure. Abundance of proinflammatory cytokine messages was measured by RNase protection assay. Exposure to LPS followed by ozone induced an inflammatory response in 4-day-old mice, which was not detected after LPS or ozone exposure alone. This exposure sequence also generated a synergistic increase in interleukin (IL)-6 mRNA abundance in 10- and 56-day-old mice but not in 4-day-old mice. Exposure to ozone followed by LPS inhibited IL-1alpha and IL-1beta responses in 4-, 10-, and 56-day-old mice; furthermore, this inhibitory effect was observed after 1.0 and 0.5 PPM ozone exposures. These results demonstrate that preexposure to LPS, which primarily activates inflammatory cell recruitment, can cause sensitization to a secondary stimulus. However, preexposure to ozone, which primarily damages the epithelium, inhibited proinflammatory responses. Thus it was concluded that sequential exposures to ozone and LPS resulted in responses not predicted by evaluating individual exposures during postnatal lung development.     

Carp horizontal cells in culture respond selectively to L-glutamate and its agonists.

Horizontal cells were enzymatically isolated from the carp retina and maintained in culture for 2-7 days. Cultured horizontal cells typically had resting membrane potentials of -50 to -70 mV and input resistances of 100-150 m omega. The cells were treated with a number of neurotransmitter agents and their analogues. Significant responses were evoked only by 3,4-dihydroxyphenylalanine (dopamine), L-glutamate, and certain glutamate analogues. The responses to dopamine were inconsistent; most often, the membrane hyperpolarized and input resistances increased. However, highly characteristic responses to L-glutamate and its analogues, quisqualate and kainate, were observed in virtually all of the cells tested. The responses consisted of an initial graded depolarization accompanied by a resistance increase, followed in most cases by a prolonged (1- to 2-min) regenerative depolarization. The regenerative component of the response appears to be Ca2+ dependent, while the underlying graded potential may be due to a decrease in K+ conductance of the membrane.     

Vasodilators inhibit acute alpha 1-adrenergic receptor-induced trophic responses in the vasculature.

Cardiovascular hypertrophy plays an important role in the development and maintenance of hypertension. Hyperactivity of the sympathetic nervous system may be one of the initiating factors responsible for the stimulation of growth processes involved in these structural alterations. We have used a well-established early biochemical marker of cellular growth processes, induction of ornithine decarboxylase (ODC), to determine whether alpha 1-adrenergic receptor-induced vascular trophic responses are dependent on arterial pressure elevation. Hydralazine or felodipine were coadministered to control the alpha 1-adrenergic receptor agonist-induced rise in mean arterial pressure (MAP). Methoxamine (2, 5, or 10 mg/kg s.c.) increased the average MAP (up to 20 mm Hg) and vascular ODC activity (up to ninefold) above control rats over 4 hours. Concomitant administration of hydralazine (0.5, 1.25, or 5 mg/kg s.c.) or felodipine (100 or 250 micrograms/kg s.c.) with methoxamine (10 mg/kg) attenuated the alpha 1-adrenergic receptor-induced activation of ODC in the aorta and mesenteric resistance vasculature, as well as the MAP increases. Vasodilators alone did not lower basal vascular ODC activity. The major findings include: 1) alpha 1-adrenergic receptor activation dose-dependently induces vascular ODC activity concomitantly with MAP elevation, 2) vasodilators inhibited both the alpha 1-adrenergic receptor-induced MAP increases and the activation of mesenteric vascular and aortic ODC, and 3) the stimulus-response correlation between MAP elevation and mesenteric (r = 0.78) and aortic (r = 0.92) ODC activation was characterized by a logistic function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of ES-62 to inhibit T-helper type 1 responses to other filarial nematode antigens

ES-62 is a secreted protein of filarial nematodes that possesses multiple immunomodulatory activities. A full characterization of these activities awaits elucidation but to date it has been shown that ES-62 can inhibit pro-inflammatory/Th1 immune responses and in some studies, it has been found to actively support Th2 development. As an active filarial nematode infection is associated with a Th2-like immunological phenotype, this study investigated whether ES-62 was likely to be responsible for, or at least contribute to, this phenotype. Specifically, we determined ES-62's effect on the immune response to two other filarial nematode antigens, chosen for their ability to promote Th1 responses. The two antigens were recombinant Onchocerca volvulus-Fatty acid And Retinol-binding-1 (rOv-FAR-1) and recombinant Onchocerca volvulus-Activation associated Secreted Protein-1 (Ov-ASP-1). Overall the results show that in spite of its previously characterized immunomodulatory properties, ES-62 was unable to modulate/reverse the Th1 immune responses induced by the two Onchocerca antigens. Therefore, in this study no support is provided for the idea that ES-62 might be a major player in facilitating the overall immunological phenotype in filariasis and reasons for this somewhat surprising outcome are discussed.     

Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia

Although kainate receptors play important roles in ischemic stroke, the molecular mechanisms underlying postischemic regulation of kainate receptors remain unclear. In this study we demonstrate that Src family kinases contribute to the potentiation of kainate receptor function. Brain ischemia and reperfusion induce rapid and sustained phosphorylation of the kainate receptor subunit GluK2 by Src in the rat hippocampus, implicating a critical role for Src-mediated GluK2 phosphorylation in ischemic brain injury. The NMDA and kainate receptors are involved in the tyrosine phosphorylation of GluK2. GluK2 binds to Src, and the tyrosine residue at position 590 (Y590) on GluK2 is a major site of phosphorylation by Src kinases. GluK2 phosphorylation at Y590 is responsible for increases in whole-cell currents and calcium influx in response to transient kainate stimulation. In addition, GluK2 phosphorylation at Y590 facilitates the endocytosis of GluK2 subunits, and the activation of JNK3 and its substrate c-Jun after long-term kainate treatment. Thus, Src phosphorylation of GluK2 plays an important role in the opening of kainate receptor channels and downstream proapoptosis signaling after brain ischemia. The present study reveals an additional mechanism for the regulation of GluK2-containing kainate receptors by Src family kinases, which may be of pathological significance in ischemic stroke.Kainate receptors are widely expressed in the mammalian central nervous system, particularly in the hippocampus, where they are involved in synaptic transmission (1), neuronal plasticity (2), and excitotoxic lesions (3). Overactivation of postsynaptic kainate receptor-mediated responses is associated with neurological disorders resulting from ischemic stroke (4). However, the intracellular processes responsible for the postischemic up-regulation of kainate receptors, and its molecular consequences, have not yet been elucidated.Reversible phosphorylation is one of the most common mechanisms regulating the function of receptor proteins. In particular, serine/threonine phosphorylation is important in the functional regulation of NMDA (5), AMPA (6), and kainate receptors (6–9), with tyrosine phosphorylation of NMDA receptors the most extensively studied (10, 11). There is accumulating evidence to show that tyrosine phosphorylation of NMDA receptors modulates their assembly at synapses after brain ischemia (12–15). However, less is known about the regulation of kainate receptor activity by tyrosine phosphorylation. Src is an important member of Src family kinases, the largest family of nonreceptor protein tyrosine kinases, and is highly expressed in the brain. Brain ischemia increases Src kinase activity in vulnerable brain regions, including the hippocampus (15–18), but it is not known whether Src phosphorylates kainate receptors.Kainate receptors are tetrameric glutamate-gated ion channels consisting of GluK1–GluK5 subunits, formerly known as GluR5–GluR7, KA1, and KA2, respectively. Functional kainate receptors can be expressed as homomers and heteromers of GluK1–3 subunits, whereas GluK4 and GluK5 subunits combine with GluK1–3 to form functional channels. It has been reported that GluK2-deficient mice are resistant to kainic acid-induced neuronal degeneration and seizures (19), and GluK2 knockdown protects against postischemic neuronal loss in the rat hippocampal CA1 region (20). In addition to sodium and potassium ions, GluK2-containing kainate receptors are permeable to Ca²⁺ (21, 22). Glutamate-induced intracellular Ca²⁺ ([Ca²⁺]_i) overload is a major mechanism underlying excitotoxicity and ischemic cell death. Furthermore, excessive activation of GluK2-containing kainate receptors triggers the proapoptotic JNK signal cascade, which contributes to ischemic brain damage (23, 24). These findings suggest that GluK2-containing kainate receptor-mediated responses are critical events in the induction of neuronal cell death after stroke.In this study we found that Src family kinases are involved in kainate-evoked whole-cell currents. In the vulnerable hippocampal CA1 region, GluK2 is phosphorylated on tyrosine 590 (Y590) by Src family kinases after brain ischemia. Conversely, the mutation of the Y590 residue on GluK2 decreases whole-cell peak currents and [Ca²⁺]_i increases elicited by kainate, and deficiency of GluK2 phosphorylation at Y590 attenuates the endocytosis of GluK2 subunits and JNK3–c-Jun activation in response to kainate. These data indicate that Src-mediated phosphorylation promotes the opening of GluK2-containing kainate receptor channels and facilitates GluK2–JNK3 signaling. Our results contribute to the elucidation of molecular mechanisms underlying brain ischemic excitotoxicity.     

Brain L-glutamate decarboxylase: purification and subunit structure.

Glutamate decarboxylase (GDCase; L-glutamate-1-carboxy-lyase, EC 4.1.1.15) was purified from whole rat brain approximately equal to 1300-fold to apparent homogeneity with a specific activity of 2.4 units per mg of protein by a combination of column chromatographies on DEAE-cellulose, hydroxylapatite, and gel filtration, and preparative nondenaturing polyacrylamide gel electrophoresis. The purified preparation contained a single protein band that comigrated with GDCase activity in three diverse analyses: nondenaturing regular (5%) and gradient (3.6-25%) polyacrylamide gel electrophoresis and isoelectric focusing at pH 4-7. The native molecular mass was calculated to be 120 +/- 10 kDa from gradient polyacrylamide gel electrophoresis and 110 +/- 10 kDa from gel filtration. Under the treatment with NaDodSO4 and 2-mercaptoethanol, GDCase dissociated into two subunits of 40 +/- 2 and 80 +/- 4 kDa, as estimated from NaDodSO4 gel electrophoresis. However, only a 40-kDa subunit was detected when GDCase was treated with 4 M urea plus NaDodSO4 and 2-mercaptoethanol, suggesting that the 80-kDa subunit is the dimer of the 40-kDa subunit. In immunoblotting, polyclonal antibodies against GDCase reacted with both 40- and 80-kDa subunits, while monoclonal antibody reacted with only 80-kDa subunits. The isoelectric point of the native enzyme was 5.4. The Km for glutamate was 1.59 X 10(-3) M. In addition to L-glutamate, cysteine sulfinic acid was also decarboxylated at approximately equal to 10% of the rate of glutamate. The pH optimum was fairly broad, with a maximum at approximately equal to 7.3. The enzyme was strongly inhibited by carbonyl-trapping agents, sulfhydryl reagents, thiol compounds, and beta-methylene-DL-aspartate.     

Abnormal retinal artery responses to stress in patients with type I diabetes

The brachial artery pressure and retinal artery pressure responses to a one-minute cold pressor test were evaluated simultaneously in 14 patients with type I diabetes mellitus (six with and eight without diabetic retinopathy) and 10 age-matched control subjects. Five patients with type I diabetes had autonomic neuropathy. Mean baseline brachial artery pressure and retinal artery pressure were similar in patients with type I diabetes and control subjects. After cold pressor testing, the brachial artery pressure increased significantly (p less than 0.01) compared with baseline values in both groups. Retinal mean arterial pressures increased significantly (p less than 0.001) after cold pressor testing compared with the baseline values only in patients with type I diabetes. Positive correlation was found between the brachial and retinal mean arterial pressures after cold pressor testing (r = 0.48; p less than 0.05) in the diabetic patients but not in the control subjects (r = 0.10; p = NS). No correlation was found between the retinal artery pressure and age of onset of diabetes, duration of diabetes, the presence or absence of diabetic retinopathy, and glycemic control. Four patients with autonomic neuropathy and low retinal artery pressures, which remained unchanged after cold pressor testing, had no diabetic retinopathy. The fifth patient with autonomic neuropathy and exaggerated systolic brachial artery pressure (175 mm Hg) and retinal artery pressure (more than 80 mm Hg) responses had severe background diabetic retinopathy. In conclusion, abnormal retinal artery responses to stress are present in patients with type I diabetes. This may be modified by the presence or absence of both autonomic neuropathy and hypertension. The biologic significance of these findings is yet to be determined.     

N-methyl-D-aspartate activates different channels than do kainate and quisqualate.

In the mammalian central nervous system, the excitatory amino acid transmitter L-glutamate activates three pharmacologically distinguishable receptors, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. The present paper addresses the issue of whether these three receptors operate independent channels or whether they share channels that may have several conductance substates. The Xenopus oocyte provides a system for expression of exogenous mRNAs that permits detailed study of receptor structure and function. In oocytes injected with rat brain mRNA, NMDA has a stoichiometry of channel activation different from that for kainate and quisqualate. NMDA activates its own channels as indicated by simple summation or near-summation of currents evoked by NMDA with those evoked by quisqualate or kainate. Deviations from summation are ascribable to lack of selectivity in which an agonist at one receptor acts as a weak antagonist at another receptor. A further indication of separate channels is that block of NMDA channels by Mg2+ or phencyclidine has no effect on kainate or quisqualate responses evoked during the block. Interactions of kainate and quisqualate are more complex, but they can be explained by lack of complete specificity of these agonists for their own receptors.     

Visual responses of neurons in somatosensory cortex of hamsters with experimentally induced retinal projections to somatosensory thalamus.

These experiments investigate the capacity of thalamic and cortical structures in a sensory system to process information of a modality normally associated with another system. Retinal ganglion cells in newborn Syrian hamsters were made to project permanently to the main thalamic somatosensory (ventrobasal) nucleus. When the animals were adults, single unit recordings were made in the somatosensory cortices, the principal targets of the ventrobasal nucleus. The somatosensory neurons responded to visual stimulation of distinct receptive fields, and their response properties resembled, in several characteristic features, those of normal visual cortical neurons. In the visual cortex of normal animals and the somatosensory cortex of operated animals, the same functional categories of neurons occurred in similar proportions, and the neurons' selectivity for the orientation or direction of movement of visual stimuli was comparable. These results suggest that thalamic nuclei or cortical areas at corresponding levels in the visual and somatosensory pathways perform similar transformations on their inputs.     

Helminth-derived products inhibit the development of allergic responses in mice

RATIONALE: Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice. OBJECTIVES: To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma. METHODS: Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung. MAIN RESULTS: The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses. CONCLUSION: Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.     

Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) recombinant DNA clones in the mouse brain cDNA library. One of the clones was positively identified as a GAD clone on the basis of the following results: (i) the clone and its secondary and tertiary clones all reacted strongly with anti-GAD antibodies; (ii) the fusion protein obtained from lambda GAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and gamma-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of approximately 2.6 kilobases that has one internal EcoRI site. After GAD cDNA was cut at the EcoRI site, two DNA fragments of about 1.6 and 1.0 kilobases were obtained at the 5' and 3' ends, respectively. The cDNA insert was found to be composed of 2632 base pairs, the translation initiation site was assigned to the methionine codon ATG, and the termination site was found to be TGA (positions 2216-2218). Furthermore, the coding region in 2169 base pairs was found to consist of 723 amino acids. The protein has a molecular weight of 83,207 and contains 83 strongly basic, 108 strongly acidic, 226 hydrophobic, and 221 polar amino acids with an isoelectric point of 5.355. The relationship of this GAD cDNA to other forms of GAD is discussed.