Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

Metastatic melanoma to the ovary in pregnancy: A case report.

Metastatic melanoma to the ovary is an uncommon presentation. We report a case of metastatic melanoma to the ovary that presented as a growing left adnexal mass during pregnancy and was thought to be benign by imaging and frozen section pathology. Here we discuss the challenges in radiologic and pathologic diagnosis, as well as considerations for the mother and newborn.

Modulation of Ca2+ channels by protein kinase C in rat central and peripheral neurons: disruption of G protein-mediated inhibition.

Activation of protein kinase C (PKC) reduced G protein-dependent inhibition of Ca2+ channels by glutamate, GA-BAB, adenosine, muscarinic, alpha-adrenergic, and LHRH receptors in a variety of central and peripheral neurons. PKC stimulation also relieved the inhibitory effect of internal GTP gamma S and reduced tonic G protein-mediated inhibition observed with internal GTP in the absence of transmitter receptor agonist. Basal Ca2+ channel currents were enhanced by PKC stimulation in most neurons studied. The PKC-induced enhancement of basal current was voltage dependent, and enhanced currents displayed altered kinetics. Inhibition of G proteins with GDP beta S attenuated the PKC-induced enhancement of basal Ca2+ channel current. These results show that PKC regulates the inhibitory effects of G proteins, possibly by disrupting the coupling of G proteins to Ca2+ channels. The PKC-induced enhancement of Ca2+ channel current results, at least in part, from the removal of tonic G protein-mediated inhibition.

Hanatoxin modifies the gating of a voltage-dependent K+ channel through multiple binding sites.

We studied the mechanism by which Hanatoxin (HaTx) inhibits the drk1 voltage-gated K+ channel. HaTx inhibits the K+ channel by shifting channel opening to more depolarized voltages. Channels opened by strong depolarization in the presence of HaTx deactivate much faster upon repolarization, indicating that toxin bound channels can open. Thus, HaTx inhibits the drk1 K+ channel, not by physically occluding the ion conduction pore, but by modifying channel gating. Occupancy of the channel by HaTx was studied using various strength depolarizations. The concentration dependence for equilibrium occupancy as well as the kinetics of onset and recovery from inhibition indicate that multiple HaTx molecules can simultaneously bind to a single K+ channel. These results are consistent with a simple model in which HaTx binds to the surface of the drk1 K+ channel at four equivalent sites and alters the energetics of channel gating.

Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels.

Hanatoxin (HaTx) binds to multiple sites on the surface of the drk1 voltage-gated K+ channel and modifies channel gating. We set out to identify channel residues that contribute to form these HaTx binding sites. Chimeras constructed using the drk1 and shaker K+ channels suggest that the S3-S4 linker may contain influential residues. Alanine scanning mutagenesis of the region extending from the C terminal end of S3 through S4 identified a number of residues that likely contribute to form the HaTx binding sites. The pore blocker Agitoxin2 and the gating modifier HaTx can simultaneously bind to individual K+ channels. These results suggest that residues near the outer edges of S3 and S4 form the HaTx binding sites and are eccentrically located at least 15 A from the central pore axis on the surface of voltage-gated K+ channels.

An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula.

The Kv2.1 voltage-activated K+ channel, a Shab-related K+ channel isolated from rat brain, is insensitive to previously identified peptide inhibitors. We have isolated two peptides from the venom of a Chilean tarantula, G. spatulata, that inhibit the Kv2.1 K+ channel. The two peptides, hanatoxin1 (HaTx1) and hanatoxin2 (HaTx2) are unrelated in primary sequence to other K+ channel inhibitors. The activity of HaTx was verified by synthesizing it in a bacterial expression system. The concentration dependence for both the degree of inhibition at equilibrium (Kd = 42 nM) and the kinetics of inhibition (kon = 3.7 x 10(4) M-1s-1; koff = 1.3 x 10(-3) s-1), are consistent with a bimolecular reaction between HaTx and the Kv2.1 K+ channel. Shaker-related, Shaw-related, and eag K+ channels were relatively insensitive to HaTx, whereas a Shal-related K+ channel was sensitive. Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx. HaTx introduces a new class of K+ channel inhibitors that will be useful probes for studying K+ channel structure and function.

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

Ion channel expression by white matter glia: the O-2A glial progenitor cell.

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.

Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin.

The biophysical properties of T-type voltage-gated calcium channels are well suited to pacemaking and to supporting calcium flux near the resting membrane potential in both excitable and non-excitable cells. We have identified a new scorpion toxin (kurtoxin) that binds to the alpha 1G T-type calcium channel with high affinity and inhibits the channel by modifying voltage-dependent gating. This toxin distinguishes between alpha 1G T-type calcium channels and other types of voltage-gated calcium channels, including alpha 1A, alpha 1B, alpha 1C and alpha 1E. Like the other alpha-scorpion toxins to which it is related, kurtoxin also interacts with voltage-gated sodium channels and slows their inactivation. Kurtoxin will facilitate characterization of the subunit composition of T-type calcium channels and help determine their involvement in electrical and biochemical signaling.

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels.

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.

Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels.

The voltage-sensing domains in voltage-gated K(+) channels each contain four transmembrane (TM) segments, termed S1 to S4. Previous scanning mutagenesis studies suggest that S1 and S2 are amphipathic membrane spanning alpha-helices that interface directly with the lipid membrane. In contrast, the secondary structure of and/or the environments surrounding S3 and S4 are more complex. For S3, although the NH(2)-terminal part displays significant helical character in both tryptophan- and alanine-scanning mutagenesis studies, the structure of the COOH-terminal portion of this TM is less clear. The COOH terminus of S3 is particularly interesting because this is where gating modifier toxins like Hanatoxin interact with different voltage-gated ion channels. To further examine the secondary structure of the COOH terminus of S3, we lysine-scanned this region in the drk1 K(+) channel and examined the mutation-induced changes in channel gating and Hanatoxin binding affinity, looking for periodicity characteristic of an alpha-helix. Both the mutation-induced perturbation in the toxin-channel interaction and in gating support the presence of an alpha-helix of at least 10 residues in length in the COOH terminus of S3. Together with previous scanning mutagenesis studies, these results suggest that, in voltage-gated K(+) channels, the entire S3 segment is helical, but that it can be divided into two parts. The NH(2)-terminal part of S3 interfaces with both lipid and protein, whereas the COOH-terminal part interfaces with water (where Hanatoxin binds) and possibly protein. A conserved proline residue is located near the boundary between the two parts of S3, arguing for the presence of a kink in this region. Several lines of evidence suggest that these structural features of S3 probably exist in all voltage-gated ion channels.

Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel.

Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins.

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.

Mental disorders and the incidence of migraine headaches in a community sample: results from the Baltimore Epidemiologic Catchment area follow-up study.

BACKGROUND: The cross-sectional relation between migraine headaches and affective disorders has been demonstrated in studies of clinical and community populations. Few studies have investigated the prospective relation between psychiatric disorders and migraine headaches. METHODS: A prospective follow-up of the Baltimore, Md, cohort of the Epidemiologic Catchment Area Study assessed psychopathologic features in 1981 and again between 1993 and 1996. Interviews included a history of headaches at baseline and self-reported assessment of migraine headaches at follow-up. Risk estimates for incident migraine headaches by 1981 demographic variables and psychopathologic features were calculated. The cross-sectional association between prevalent migraine and lifetime psychiatric diagnoses was estimated. RESULTS: In the at-risk population of 1343, there were 118 incident cases of migraine headaches. The age- and sex-specific incident rates of migraine headaches followed the expected patterns, with younger age and female sex identified as risk factors. In cross-sectional analyses, major depression (odds ratio, 3.14; 95% confidence interval, 2.03-4. 84) and panic disorder (odds ratio, 5.09; 95% confidence interval, 2. 65-9.79) had the strongest associations, and alcohol and other substance abuse were not associated. In logistic regression models including age, sex, and psychiatric illness in 1981, only phobia was predictive of incident migraines (odds ratio, 1.70; 95% confidence interval, 1.11-2.58). Affective disorders were not predictive of incident migraine headaches. Including a history of tricyclic antidepressant use did not change the results. CONCLUSIONS: There is a strong cross-sectional relation between affective disorders and migraine headaches in this cohort. However, there is no association between antecedent affective disorders and incident migraine headaches in this population-based prospective study.

Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid. Effect of tryptophan loading.

Extracellular fluid levels of the neurotoxin quinolinic acid in the corpus striatum of rats, measured by in vivo microdialysis, were increased in a dose-dependent manner following the intraperitoneal administration of tryptophan. The lowest dose of tryptophan (12.5 mg/kg), equivalent to about 5% of the normal daily intake, increased peak quinolinic acid levels nearly 3-fold. At higher doses of tryptophan (up to 250 mg/kg), concentrations of quinolinic acid increased over 200-fold and exceeded potentially neurotoxic levels (10 microM). In contrast, the increase in extracellular serotonin following even the highest tryptophan dose was small (less than 2-fold). These data indicate that quinolinic acid is present in the extracellular fluid where it may function as a neuromodulator and that it is very responsive to physiological changes in precursor availability.

Kynurenine metabolites of tryptophan: implications for neurologic diseases.

Over the past 2 decades, a number of studies have demonstrated that amino acids act as precursors for the biosynthesis of a variety of neuroactive compounds, including catecholamines and indoleamines. For example, the aromatic amino acid L-tryptophan is a precursor for serotonin biosynthesis. Based on this observed precursor relationship, dietary tryptophan supplementation is used to treat a number of neurologic disorders attributed to alterations in serotoninergic neurotransmission. Recent studies have revealed that, in addition to serotonin, a number of neuroactive compounds, the kynurenines, are metabolities of tryptophan. Of these, perhaps the most important is quinolinic acid, a neurotoxin that acts at the N-methyl-D-aspartate (NMDA) receptor and whose precursor responsiveness to tryptophan far exceeds that of serotonin. In the central nervous system, kynurenines, and in particular quinolinic acid, may modulate excitatory amino acid transmission, and may act as neurotoxic agents implicated in the pathogenesis of several neurologic diseases.

Immune induction of human monocyte plasminogen activator. Characteristics of an assay for cell-mediated immunity.

We characterized immunologic induction of monocyte plasminogen activator (PA) to determine whether assay for PA induction reliably detected cell-mediated immunity (CMI). Mononuclear leukocytes (MNL) were incubated in teflon-lined culture tubes for 1-4 days in the presence or absence of phytohemagglutinin-P (PHA), concanavalin A (Con A) or Candida antigen. PA activity of the monocytes in those suspensions was then measured using a micro fibrin plate assay. Monocytes in stimulated MNL had more PA activity than monocytes in unstimulated MNL. Maximal differences between stimulated and unstimulated cells were seen after 2 days of culture. Dose-response studies demonstrated that PA induction occurred at submitogenic concentrations of stimuli. Peak induction was seen using suboptimally mitogenic concentrations of PHA, Con A and Candida antigen. PA induction in response to Candida stimulation corresponded with skin test results. More than 90% of healthy adults tested had positive assays to all stimuli. LPS, in picogram concentrations, induced PA activity in the absence of lymphocytes, but such induction was prevented by polymyxin B. Supernates from activated MNL also induced PA in purified monocytes. This indirect assay of PA induction was less sensitive than direct assay of the MNL. A standard indirect assay for leukocyte inhibitory factor (LIF) was also less sensitive than the direct PA induction assay. The direct PA induction assay is sensitive and convenient and requires small volumes of blood. It may prove valuable in in vitro analysis of cell-mediated immunity in health and disease.

Characterization of monoclonal antibodies against rat glandular kallikrein.

Monoclonal antibodies can be produced in large amounts, are homogenous and can be highly purified. A specific monoclonal antibody against glandular kallikrein could be very useful in studies of the kallikrein-kinin system, both in vivo and in vitro. Two monoclonal antibodies against rat glandular kallikrein (rgKK) were produced by immunized mouse spleen and lymph node fusion with myeloma Ag8.653. Both antibodies, named 2E9.8 and 2E9.9, bound active 125I-kallikrein and phenylmethylsulfonyl fluoride (PMSF)-inactivated 125I-kallikrein. A radioimmunoassay (RIA) was developed with each of the antibodies using rabbit anti-mouse gamma globulin to separate bound from free 125I-rgKK. The standard curve (range 10-1000 ng/tube) was curved even when subjected to logit-log transformation. Using 3% polyethylene glycol (PEG) to assist separation of bound from free, the standard curve became straight for 2E9.8 and the RIA was more sensitive, with a binding range of 0.35-2.4 ng/tube. Both antibodies were specific for rgKK since they had negligible cross-reaction with purified proteases from the submandibular gland of the rat (tonin, esterases B and E). They did not cross-react with mouse nerve growth factor, epidermal growth factor, nor with pig pancreatic kallikrein. Antibody 2E9.9 did appear to bind some human kallikrein when tested with high concentrations of this enzyme, while 2E9.8 did not. When preincubated with purified rgKK, both antibodies prevented the enzyme from releasing kinins from semi-purified dog kininogen and from cleaving [3H]-L-arginine methyl ester (3H-TAME). These results suggested that both antibodies bind an epitope near to, and maybe including, the active site of the enzyme. Monoclonal antibody 2E9.8 appears to be specific for rgKK, can be used in a sensitive RIA, and is capable of inhibiting the enzymatic activity of kallikrein. It should prove to be useful in vivo for examining the role of kallikrein in physiological processes.

Effects of sulfur-containing analogues of stearic acid on growth and fatty acid biosynthesis in the protozoan Crithidia fasciculata.

A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations. The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration. The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived.