Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Heterogeneity at the HLA-DRB1 allelic variation locus does not influence multiple sclerosis disease severity, brain atrophy or cognition.

BACKGROUND: HLA-DRB1*1501 (DR15) and other HLA class II alleles increase the risk of developing multiple sclerosis (MS). However, the contribution of genetic heterogeneity to the clinical course of MS remains controversial. We examined the influence of DR15 and other common DRB1 alleles (DRB1*01 (DR1), DRB1*03 (DR3) and DRB1*04 (DR4) on MS severity in a large, Australian, population-based cohort. METHODS: We studied the association between common HLA-DRB1 alleles and genotypes and age of onset as well as three clinical disease severity descriptors: Multiple Sclerosis Severity Score, progression index), and the interval between the first and second attack in 978 patients with relapsing remitting MS and secondary progressive MS. We assessed cognition using the Symbol Digit Modalities Test in 811 patients and brain atrophy using the linear magnetic resonance imaging marker, the intercaudate ratio, in 745 patients. RESULTS: Carrying DR15 significantly decreased the age of MS onset by 3.2 years in homozygotes and 1.3 years in heterozygotes. Carrying the HLA-DR15, -DR1, -DR3 or -DR4 alone or in combination did not affect clinical disease severity, cognition or cerebral atrophy. CONCLUSIONS: This study confirms that heterogeneity of HLA-DRB1 does not influence disease outcome in relapsing MS patients, with the exception of a younger age of onset in HLA-DR15 carriers.

Resistance to Leishmania major is linked to the H2 region on chromosome 17 and to chromosome 9.

In Leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. Here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. Mice infected with L. major show marked genetic differences in disease manifestations: BALB/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas C57BL/6 are resistant, developing small, self-healing lesions. F2 animals from a C57BL/6 X BALB/c cross showed a continuous distribution of lesion score. Quantitative trait loci (QTL) have been mapped after a non-parametric QTL analysis on a genome-wide scan on 199 animals. QTLs identified were confirmed in a second cross of 271 animals. Linkage was confirmed to a chromosome 9 locus (D9Mit67-D9Mit71) and to a region including the H2 locus on chromosome 17. These have been named Imr2 and Imr1, respectively.

HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis.

Human leucocyte antigen (HLA)-DRB1*1501 and other class II alleles influence susceptibility to multiple sclerosis (MS), but their contribution if any to the clinical course of MS remains uncertain. Here, we have investigated DRB1 alleles in a large sample of 1230 Australian MS cases, with some enrichment for subjects with primary progressive (PPMS) disease (n = 246) and 1210 healthy controls. Using logistic regression, we found that DRB1*1501 was strongly associated with risk (P = 7 x 10(-45)), as expected, and after adjusting for DRB1*1501, a predisposing effect was also observed for DRB1*03 (P = 5 x 10(-7)). Individuals homozygous for either DRB1*15 or DRB1*03 were considerably more at risk of MS than heterozygotes and non-carriers. Both the DRB1*04 and the DRB1*01/DRB1*15 genotype combination, respectively, protected against PPMS in comparison to subjects with relapsing disease. Together, these data provide further evidence of heterogeneity at the DRB1 locus and confirm the importance of HLA variants in the phenotypic expression of MS.

The human Y chromosome: overlapping DNA clones spanning the euchromatic region.

The human Y chromosome was physically mapped by assembling 196 recombinant DNA clones, each containing a segment of the chromosome, into a single overlapping array. This array included more than 98 percent of the euchromatic portion of the Y chromosome. First, a library of yeast artificial chromosome (YAC) clones was prepared from the genomic DNA of a human XYYYY male. The library was screened to identify clones containing 160 sequence-tagged sites and the map was then constructed from this information. In all, 207 Y-chromosomal DNA loci were assigned to 127 ordered intervals on the basis of their presence or absence in the YAC's, yielding ordered landmarks at an average spacing of 220 kilobases across the euchromatic region. The map reveals that Y-chromosomal genes are scattered among a patchwork of X-homologous, Y-specific repetitive, and single-copy DNA sequences. This map of overlapping clones and ordered, densely spaced markers should accelerate studies of the chromosome.

The human Y chromosome: a 43-interval map based on naturally occurring deletions.

A deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. The individuals studied included XX males, XY females, and persons in whom chromosome banding had revealed translocated, deleted, isodicentric, or ring Y chromosomes. Most of the 132 Y chromosomal loci mapped were sequence-tagged sites, detected by means of the polymerase chain reaction. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals, all defined by naturally occurring chromosomal breakpoints and averaging less than 800 kilobases in length. This deletion map should be useful in identifying Y chromosomal genes, in exploring the origin of chromosomal disorders, and in tracing the evolution of the Y chromosome.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Replication of KIAA0350, IL2RA, RPL5 and CD58 as multiple sclerosis susceptibility genes in Australians.

A recent genome-wide association study (GWAS) conducted by the International Multiple Sclerosis Genetics Consortium (IMSGC) identified a number of putative MS susceptibility genes. Here we have performed a replication study in 1134 Australian MS cases and 1265 controls for 17 risk-associated single nucleotide polymorphisms (SNPs) reported by the IMSGC. Of 16 SNPs that passed quality control filters, four, each corresponding to a different non-human leukocyte antigen (HLA) gene, were associated with disease susceptibility: KIAA0350 (rs6498169) P=0.001, IL2RA (rs2104286) P=0.033, RPL5 (rs6604026) P=0.041 and CD58 (rs12044852) P=0.042. There was no association (P=0.58) between rs6897932 in the IL7R gene and the risk of MS. No interactions were detected between the replicated IMSGC SNPs and HLA-DRB1*15, gender, disease course, disease progression or age-at-onset. We used a novel Bayesian approach to estimate the extent to which our data increased or decreased evidence for association with the six most-associated IMSGC loci. These analyses indicated that even modest P-values, such as those reported here, can contribute markedly to the posterior probability of 'true' association in replication studies. In conclusion, these data provide support for the involvement of four non-HLA genes in the pathogenesis of MS, and combined with previous data, increase to genome-wide significance (P=3 x 10(-8)) evidence of an association between KIAA0350 and risk of disease.

Genetic absence epilepsy rats from Strasbourg have increased corticothalamic expression of stargazin.

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.

Chromosomes of malaria parasites.

The advent of pulsed field gradient electrophoresis has proved remarkably useful for studying chromosomes of the genetically intractable malaria parasite Plasmodium falciparum. Advances include determination of the karyotype, a linkage map and restriction maps of individual chromosomes that enable the ordering of genes. The structural basis underlying a frequently occurring form of chromosome size polymorphism is now understood and other polymorphisms are providing tantalizing clues to the mechanisms underlying drug resistance.

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Identification of a genetic locus modulating splenomegaly induced by granulocyte colony-stimulating factor in mice.

Clinically detectable splenomegaly and splenic rupture are uncommon but potentially life-threatening consequences of G-CSF administration. Increased spleen size in mice injected with G-CSF is a complex genetic trait amenable to investigation in experimental inter-strain crosses by quantitative trait analysis. A quantitative trait locus (QTL) with highly significant linkage (LOD 7.9) for splenomegaly was identified within a 22 centimorgan (cM) region on chromosome 1. Inheritance of a C57BL/6 haplotype in this region was associated with a greater spleen weight. The relevance of this locus was confirmed by analysing the responses of mice congenic for the distal 12 cM of this region (C57BL/6 and C57BL/6.SJL-Ptprc(a) Pep3(b)). Consistent with the QTL effect, mice lacking C57BL/6 alleles in this region had reduced splenomegaly induced by G-CSF. Intriguingly, peripheral blood neutrophilia and progenitor cell mobilisation responses to G-CSF were also significantly influenced.

Human chorionic cells in primary culture: a model for renin biosynthesis.

Amnion and chorion cells from human fetal membranes have been cultured. Chorionic cells secrete renin whereas amnionic cells do not. Renin is secreted by chorionic cells as an inactive form that can be activated by trypsin treatment or acid dialysis. The antigenic and enzymatic properties of activated chorionic renin and kidney renin are similar. Incorporation of [35S]methionine in the culture demonstrates that chorionic renin is secreted as a high molecular weight form, 54K. This 54K inactive renin could represent the proenzyme.

Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients.

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.

Noradrenergic and serotoninergic innervation of cortical, thalamic, and tectal visual structures in Old and New World monkeys.

Antisera directed against human dopamine-beta-hydroxylase and against serotonin were used to characterize the noradrenergic (NA) and serotoninergic (5-HT) innervation of several cortical and subcortical visual areas in squirrel monkey (Saimiri sciureus) and cynomolgus monkey (Macaca fascicularis). Few species differences were observed for either monoamine. Cortical areas 17 and 18, as well as visual areas in the temporal and parietal lobe were found to exhibit regional specialization of both 5-HT and NA innervation. Precisely at the border between areas 17 and 18, the laminar innervation patterns and density characteristic of NA fibers in area 17 (Morrison et al., '82a; Kosofsky et al., '84) shift so that layer IV of area 18 contains more fibers than layer IV of area 17, and the overall density of fibers in area 18 is higher. For 5-HT, the highly laminated patterns characteristic of area 17 (Morrison et al., '82a; Kosofsky et al., '84) also observe this cytoarchitectonic boundary. Fibers in area 18 are more evenly distributed across laminae, and the overall density of fibers decreases. The visual region of the inferotemporal cortex was found to be very lightly innervated by NA fibers and very densely innervated by 5-HT fibers. Area 7 of the parietal lobule was more densely innervated by NA fibers, and less densely innervated by 5-HT fibers, than any other visual cortical region examined. The visual thalamic nuclei exhibited even greater regional differences in the density of NA innervation. The lateral geniculate nucleus was found to be virtually devoid of NA fibers, while the pulvinar-lateral posterior complex was densely innervated. The density of 5-HT fibers was more uniform across thalamic visual nuclei. The lateral geniculate, pulvinar, and lateral posterior nuclei all exhibit a moderate to high density of immunoreactive fibers. In the mesencephalon, the superficial layers of the superior colliculus were found to be densely innervated by NA fibers, whereas 5-HT fibers were most dense in the intermediate layers. These patterns of innervation indicate that, in these primate species, functionally related visual regions share common and distinguishable densities of NA innervation. Specifically, tecto-pulvinar-juxtastriate structures are more densely innervated than geniculo-striate and inferotemporal structures. These relationships suggest that, within the visual system, NA fibers preferentially innervate the regions involved in spatial analysis and visuomotor response rather than those involved in feature extraction and pattern analysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of corticotropin-releasing-factor-like immunoreactivity in brainstem of two monkey species (Saimiri sciureus and Macaca fascicularis): an immunohistochemical study.

Immunohistochemical methods were utilized to systematically map the distribution of corticotropin-releasing-factor-like immunoreactivity (CRF-LI) in the diencephalon, mesencephalon, and rhombencephalon of two monkey species (Saimiri sciureus and Macaca fascicularis). A primary antiserum directed against the human form of the peptide was utilized. Immunoreactive neuronal perikarya and processes were evident in numerous areas, and the distributions of these elements were similar for the two species. As previously reported for rats, monkeys, and human, intense immunoreactivity was evident in putative hypophyseal neurons in the parvicellular component of the paraventricular nucleus of the hypothalamus and in fibers extending from this area into the median eminence. The results for other brainstem regions, most of which have been previously examined for CRF-LI only in rats, indicate that many similarities exist between rats and monkeys in the distribution of this peptide in brainstem extrahypophyseal neuronal circuits, although substantial differences are also evident. For example, immunoreactive perikarya previously observed in other hypothalamic nuclei in rats were not evident in monkeys. Conversely, in monkeys, unlike rats, labeled perikarya were evident in several thalamic nuclei, especially in the intralaminar complex. Also, two large groups of immunoreactive neurons which have generally not been observed in rat studies were present in the mesencephalon and rhombencephalon. In the mesencephalon this consisted of a group of neurons just lateral to the mesencephalic tegmentum, extending throughout the rostral-caudal extent of the midbrain. In the rhombencephalon, labeled perikarya were observed throughout the inferior olive. Some of the differences between rats and monkeys in the locations of labeled perikarya may be due to differences in antiserum specificity and/or sensitivity, or they may result from the fact that colchicine pretreatment was not utilized in the present study. The distributions of immunoreactive fibers also exhibited similarities and differences between monkeys and rats. The most striking terminal fields observed in the present study which have not been previously described are a moderate-to-dense field within and adjacent to presumed dopamine-containing neurons in the substantia nigra pars compacta, a dense innervation of certain subdivisions of the interpeduncular nucleus, and a regionally and parasagittally organized distribution of fibers in the Purkinje cell and molecular layers of the cerebellar cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of corticotropin-releasing factor-like immunoreactivity in squirrel monkey (Saimiri sciureus) amygdala.

Previous anatomical studies of corticotropin-releasing factor (CRF)-like immunoreactivity in rat brain have reported prominent clustering of neuronal elements containing this peptide within the amygdala. The highest concentrations of both CRF-positive cells and fibers were evident in the central nucleus, an observation consistent with the putative role of this peptide in autonomic and endocrine regulation. In addition, lower densities of CRF-positive somata and processes have been noted in other amygdaloid nuclei. However, the distribution of CRF-like immunoreactivity in the amygdala has not been described for any primate species. Such a description would be of interest since substantial differences in the distribution of CRF in rodent and primate have been reported for other brain regions. The present study uses immunohistochemical methods, with a polyclonal antiserum directed against the human form of CRF, to determine the distribution of this peptide in non-colchicine-treated monkeys (Saimiri sciureus). Within the amygdaloid complex, the most numerous and concentrated collections of CRF-positive neurons were seen in the basal and lateral nuclei. The highest densities of CRF-positive fibers and terminals were seen in the lateral and central amygdaloid nuclei. Moderately dense plexuses of CRF-positive fibers also were seen in layer Ia of the periamygdaloid cortex, nucleus of the lateral olfactory tract, anterior and posterior cortical nuclei, and the medial nucleus. Thus, the distribution of CRF-like immunoreactivity differs substantially in monkey and rat amygdala. Since CRF-positive perikarya in monkey are most prominent in nuclei with pronounced interconnections with neocortex, these differences may be an integral component of the increased cortical development that characterizes the primate brain.

Histochemical characterization of a neocortical projection of the nucleus locus coeruleus in the squirrel monkey.

Histochemical evidence is presented for a catecholamine-containing projection from the nucleus locus coeruleus to the neocortex in the squirrel monkey. The innervation of superior temporal gyrus has been examined in particular. Glyoxylic acid-induced fluorescence shows an extensive arborization of fine, catecholamine-containing fibers with prominent varicosities in all layers of the neocortex. The nucleus locus coeruleus is identified as a source of these fibers by both ortho- and retrograde axonal tracing techniques. After injection of horseradish peroxidase into the neocortex, labelled cell bodies are localized throughout the major portions of the locus coeruleus. Conversely, after microinjection into the nucleus locus coeruleus, tritiated proline is transported into the neocortex where it appears within fibers similar in distribution to those revealed by fluorescence histochemistry. Both transport techniques indicate that cortical projections of the locus coerculeus originate from both ipsilateral and contralateral nuclei.