Gambierol acts as a functional antagonist of neurotoxin site 5 on voltage-gated sodium channels in cerebellar granule neurons.

The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 microM, gambierol alone had no effect on the intracellular Ca2+ concentration [Ca2+]i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM-10 microM). However, gambierol was a potent inhibitor (IC50 = 189 nM) of the elevation of [Ca2+]i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC50 = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca2+ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca2+]i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca2+]i in a concentration-dependent manner (IC50 = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.

The neurotoxic lipopeptide kalkitoxin interacts with voltage-sensitive sodium channels in cerebellar granule neurons.

The marine neurotoxin kalkitoxin, a thiazoline-containing lipid derived from the pantropical marine cyanobacterium Lyngbya majuscula, was assayed for interaction with the tetrodotoxin-sensitive, voltage-sensitive sodium channel (TTX-VSSC) in cerebellar granule neuron cultures (CGN). The naturally occurring isomer of kalkitoxin (KTx-7) blocked veratridine-induced (30 microM) neurotoxicity in a concentration-dependent manner (EC50 22.7 nM [9.5-53.9 nM, 95% confidence interval {CI}]) in CGN. Kalkitoxin was a potent inhibitor (EC50 26.1 nM [12.3-55.0 nM, 95% CI]) of the elevation of intracellular Ca2+ concentration [Ca2+](i) that accompanies exposure of CGN to veratridine. To further explore the potential interaction of KTx-7 with TTX-VSSC, we assessed the influence of KTX-7 on the binding of [3H]batrachotoxin ([3H]BTX) to neurotoxin site 2 on the TTX-VSSC. Although kalkitoxin was without effect on the basal binding of [3H]BTX to intact cerebellar granule neurons, in the presence of the positive allosteric modulator, deltamethrin, [3H]BTX binding was inhibited by KTx-7 in a concentration-dependent manner (11.9 nM [IC50=3.8-37.2 nM, 95% CI]). These results provide both direct and functional evidence for an interaction of kalkitoxin with the neuronal TTX-VSSC.

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors.

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.

Brevetoxin derivatives act as partial agonists at neurotoxin site 5 on the voltage-gated Na+ channel.

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid-soluble polyether neurotoxins produced by the marine dinoflagellate Karina brevis, an organism associated with 'red tide' blooms in the Gulf of Mexico. Ingestion of shellfish contaminated with K. brevis produces neurotoxic shellfish poisoning (NSP) in humans. NSP symptoms emanate from brevetoxin activation of neurotoxin site 5 on voltage-gated sodium channels (VGSC) [Toxicon 20 (1982) 457]. In primary cultures of rat cerebellar granule neurons (CGN), brevetoxins produce acute neuronal injury and death. The ability of a series of naturally occurring and synthetic brevetoxins to trigger Ca(2+) influx in CGN was explored in the present study. Intracellular Ca(2+) concentration was monitored in fluo-3-loaded CGN using a fluorescent laser imaging plate reader. The naturally occurring derivatives PbTx-1, PbTx-2 and PbTx-3 all produced a rapid and concentration-dependent increase in cytosolic [Ca(2+)]. The maximum response to PbTx-1 was approximately two-fold greater than that of either PbTx-2 or PbTx-3. Two synthetic derivatives of PbTx-3, alpha-naphthoyl-PbTx-3 and beta-naphthoyl-PbTx-3, were also tested. Both alpha- and beta-naphthoyl-PbTx-3 stimulated a rapid and concentration-dependent Ca(2+) influx that was, however, less efficacious than that of PbTx-3. These data indicate that, analogous to neurotoxin site 2 ligands, activators of neurotoxin site 5 display a range of efficacies, with PbTx-1 being a full agonist and other derivatives acting as partial agonists.

Spectral integral representations of volume scattering in sediments in layered waveguides.

In situ measurements of scattering strength are often obtained by analyzing the early-time, high-angle reverberation from bottom and subbottom features. In order to provide insight into the mechanisms which cause bottom reverberation, and to their distinguishing characteristics, it is necessary to have a capability for modeling both the rough surface and the volume scattering mechanisms. For high-angle, early-time backscatter, the most appropriate approach is to use a spectral integral representation, which naturally includes the continuous spectrum important for this angular regime. A rough surface scattering theory developed earlier in this framework has provided important insights into wave scattering and penetration physics at the seafloor. Here a consistent representation for the subbottom scattering is developed and examples are provided which illustrate the observable differences between the two scattering mechanisms.

Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas.

Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing-web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge.

Association between IgM response to IgG damaged by glyoxidation and disease activity in rheumatoid arthritis.

OBJECTIVE: To determine the association of serum IgG advanced glycation endproducts (AGE) and IgM anti-IgG-AGE antibodies with clinical measurements of rheumatoid arthritis (RA) disease activity. METHODS: The study group consisted of 62 patients with RA and 16 control patients with osteoarthritis. Patient derived variables included perceived disease activity (10 cm visual analog scale, VAS) and Health Assessment Questionnaire (HAQ) results. Clinical measures of RA activity consisted of tender and swollen joint counts and a physician evaluation of disease activity (by VAS) as well as history of nodules, bone erosions, Sjogren's syndrome, and vasculitis documented by chart review. Patient sera were evaluated for glucose, glycosylated hemoglobin, and presence of RF, IgG-AGE and IgM anti-IgG-AGE. The nitroblue tetrazolium colorimetric and aminophenyl boronic acid methods were used for measurement of IgG-AGE, along with an ELISA for measurement of IgM anti-IgG-AGE. RESULTS: Significant correlations were found between the presence of IgM anti-IgG-AGE and clinical measurements of swollen joint count and physician VAS. CONCLUSION: IgM anti-IgG-AGE appears to be associated with clinical measurements of RA activity and represents a new marker of more active disease in RA.

Advanced glycation endproducts (AGE) on IgG, a target for circulating antibodies in North American Indians with rheumatoid arthritis (RA).

Several tribes of North American Indians are known to have poor glucose control and are at a high risk of developing type 2 diabetes. Similarly some tribes also exhibit RA at a high frequency. We have recently determined that a subset of Caucasian patients with RA mount an immune response to IgG modified with advanced glycation endproducts (AGE). The AGE modifications on IgG in vivo include N(epsilon)-(carboxymethyl) lysine, imidazolone and pentosidine. The presence of IgG-AGE and the antibody response to the IgG-AGE in the Ojibwe tribe of First Nations native Indians where both NIDDM and RA are prevalent was investigated. AGE modified IgG and albumin were determined using a modified nitroblue tetrazolium assay. Rheumatoid factors (RFs) and IgM and IgA anti-IgG-AGE were detected by ELISA. Of the 108 individuals tested, 21 had RA only, 3 had both RA and type 2 diabetes, 30 had type 2 diabetes only and 51 had no diagnosed disease. AGE modified IgG was significantly elevated in the RA group compared to the diabetic group. IgM and IgA RFs were detected in 83% and 50% of the RA patients, compared to 31-37% and 7-10% of the diabetics or normal individuals. IgM anti-IgG-AGE was detected in 54% of the RA patients, in contrast to 7-14% in the diabetics or normal individuals. IgA anti-IgG-AGE was detected in 42% of the RA patients and only 7 to 8% of the NIDDM or normal individuals. The IgM or IgA anti-IgG-AGE antibodies likely contribute to the accumulation of IgG-AGE, possibly through blocked clearance through AGE receptors. A trend towards more severe disease was seen in those Ojibwe RA patients with circulating anti-AGE antibodies. Non-enzymatic glycation may be an important pathogenic link in the RA seen in North American Indians.

Antibody response to sheep red blood cells in major histocompatibility (B) complex aneuploid line of chickens.

An integral part of the immune response is the production of antibodies specific for different antigenic challenges. Genes of the MHC encode products that regulate immunity. This study utilized the FCT-15 line of chickens, which is aneuploid for the chromosome containing the ribosomal RNA genes (rDNA) and the MHC or B complex to determine whether an antibody response to SRBC would vary as a function of B complex gene dose. Mating of trisomic parents (B15B15B15) animals produced progeny having either a disomic (B15B15), trisomic (B15B15B15), or tetrasomic (B15B15B15B15) B complex dosage. The number of B/rDNA chromosomes, and thus the B complex dosage was determined by feather pulp nucleolar typing of chicks at hatch. A 5% SRBC antigenic challenge, which induces a T cell-dependent antibody response, was injected at 6 wk of age. Samples taken prior to SRBC injection as well as 5, 8, and 12 d postinjection were assayed for total and mercaptoethanol-resistant antibody. Peak antibody titers (log2), day of peak titer and rate of titer decline were calculated using a quadratic equation for each bird. Differences among the three B complex dosages were evaluated by analysis of variance. Antibody titers rose from 5 to 8 d postinjection and declined thereafter without significant differences among the three B complex doses. Calculations from the quadratic equations showed that B complex dose affected neither peak antibody titer nor day of peak titer. However, trisomic and tetrasomic animals had significantly more rapid rates of decline from the maximum titer. In aneuploid chickens, changes in antigen processing, antigen presentation, or persistence of processed antigen may maintain levels of antibody production found in disomic chickens and explain the more rapid decline of titer.

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

Alternating unilateral lachrymation.

A 56-year-old woman displayed a condition of alternating unilateral lachrymation while she was undergoing psychotherapy. Although she was aware of this condition only since her marriage, hypnotic age regression revealed its existence in childhood, together with mutism and a catatonic trance-like state. Further investigation revealed the connection of this symptom with her family constellation and the reappearance of the unilateral crying as an adult. In this paper we review the neurophysiology of lachrymation, discuss the hypnosis sessions, discuss the symptom as a psychosomatic condition and as a dissociative phenomenon, and present a case report of her background.