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Metabolic mapping of brain regions associated with behavioral extinction in preweanling rats.

Fluorodeoxyglucose autoradiography, quantitative image analysis, and a multivariate tool (partial least squares) were used to assess distributed patterns of brain activation in postnatal day 17 and day 12 rat pups engaged in extinction of instrumental behavior. Pups were trained in a straight alley runway on an alternating reward schedule, or on a pseudorandom reward schedule, injected with fluorodeoxyglucose, and then shifted to continuous nonreward (extinction). Another group at each age served as handled controls. Day 17 pups trained on the alternating schedule demonstrated faster extinction rates compared to those trained on the pseudorandom schedule, a phenomenon known as the partial reinforcement extinction effect. No differences were found between day 12 groups. Partial least-squares analysis revealed age-related increases in fluorodeoxyglucose uptake across all three training conditions in the cingulate and frontal cortices, amygdala, midline thalamic nuclei, cerebellum, and in several brainstem regions. Training-related increases common to both age groups were found in the orbital frontal cortex, limbic thalamus, gigantocellular reticular nucleus, the somatosensory system, and cerebellum. Age-dependent training effects were found in the interpositus and medial cerebellar nuclei wherein fluorodeoxyglucose uptake increased in the day 12 alternation and pseudorandom groups relative to controls. Day 12 pups trained on the alternating schedule demonstrated increased uptake in the anterior dorsal thalamus relative to pseudorandom and control pups. Hence, a large-scale neural system comprised by somatosensory, cerebellar, and brainstem regions govern extinction behavior in preweanling rats. Recruitment of limbic structures may allow the older pups to modify extinction behavior based on prior learning.

Age Factors↗

The mouse T complex gene Tsga2, encoding polypeptides located in the sperm tail and anterior acrosome, maps to a locus associated with sperm motility and sperm-egg interaction abnormalities.

Previous studies of sperm from mice heterozygous for a t haplotype (t) and heterospecific combinations of the t complex identified two tightly linked genetic factors responsible for t/t male sterility related to expression of the flagellar waveform aberration, curlicue. Dnahc8, an axonemal dynein heavy chain gene, is a strong candidate for the proximal factor, Ccua, but the identity of the distal factor, Ccub, is unknown. In the present study, we employ motility assays of sperm from males heterozygous for t and novel heterospecific combinations of the t complex to demonstrate that Ccub is a composite of at least two synergic elements, Ccub1, positioned within a genomic interval spanning approximately 0.6 Mb immediately distal to Dnahc8, and Ccub2, situated in a region approximately 4-7 Mb distal to Ccub1. We also show that Tsga2, a testis-restricted gene, fulfills many of the prerequisites required to make it a strong candidate for Ccub1. These include: 1) its location within the aforementioned genomic interval; 2) a highly reduced level of testis expression by its heterospecific allele relative to the level of expression of its t allele; 3) determination that TSGA2(t) carries numerous nonsynonymous mutations in residues otherwise highly conserved in all known orthologous proteins; 4) the detection of major TSGA2 polypeptides in sperm protein extracts; and 5) the apparent distribution of these polypeptides in major sperm tail structures. Surprisingly, these TSGA2 isoforms appear to localize in the vicinity of the anterior acrosome, as well, suggesting that Tsga2 may also play a role in sperm-egg interaction. Finally, our results indicate that a TSGA2 polypeptide with apparent similarities to the smaller of the two sperm isoforms is expressed by epididymal cells.

Acrosome↗

Novel human HALR (MLL3) gene encodes a protein homologous to ALR and to ALL-1 involved in leukemia, and maps to chromosome 7q36 associated with leukemia and developmental defects.

We have identified and characterized the approximately 12-kb cDNA of a novel human gene (designated HALR for "homologous to ALR" and given the symbol MLL3 by the HUGO Gene Nomenclature Committee) for which open reading frame (ORF) encodes a predicted large hydrophilic nuclear protein comprising 4,025 amino acids with a calculated molecular mass of approximately 443 kD. Within the amino acid sequence of HALR were identified a SUVAR3-9, enhancer of zeste, trithorax (SET) domain, three plant homeodomain (PHD)-type zinc fingers, a high motility group (HMG)-1 box, a leucine-zipper-like pattern, two potential transactivating domains, several nuclear localization signals, and multiple nuclear receptor interaction signature motifs. Especially within the SET domain, PHD fingers and several other regions, the HALR protein exhibits significant similarity to ALR (acute lymphoblastic leukemia [ALL]-1 related), ALL-1/myeloid/lymphoid or mixed-lineage leukemia (ALL-1/MLL), and trithorax, evolutionarily conserved proteins that influence differentiation and development. Northern blot analysis demonstrated transcripts of approximately 11-12 kb, while reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that HALR is expressed in a wide range of human tissues and cancer cell lines. The HALR gene contains 46 exons, is estimated to span >101 kb, and is located on chromosome region 7q36. Terminal 7q deletions are common chromosomal aberrations encountered in hematological neoplasia and in holoprosencephaly 3, a midline embryonic defect involving forebrain development. We have also isolated the partial cDNA of the murine homologue of HALR, which displays high homology to its human counterpart. Taking into consideration its notable protein motifs, ubiquitous expression, evolutionary conservation and chromosomal position, HALR is likely to play a housekeeping role in transcriptional regulation, and may be involved in leukemogenesis and developmental disorders.

Amino Acid Sequence↗

Fine mapping of diabetes-associated IA-2 specific autoantibodies.

The related tyrosine phosphatase-like proteins (PTP) IA-2 and IA-2beta are autoantigens of type 1 diabetes. Autoantibodies are predominantly against IA-2. We utilized the close homology between IA-2 and IA-2beta PTP domains to design chimeras and mutants in order to identify humoral IA-2-specific epitopes. Fifteen sera with antibodies to IA-2 specific PTP domain epitopes were tested against IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033), IA-2beta(741-848)/IA-2(795-845)/IA-2beta(900-1033), and IA-2beta(741-898)/IA-2(845-875)/IA-2beta(930-1033)chimeras. Two sera bound IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033)and IA-2beta(741-848)/IA-2(795-845)/IA-2beta(900-1033)only indicating that the IA-2 specific residues 859, 862, and/or 867 were critical for antibody binding. Mutation of glutamine 862 abolished binding in one of these sera. Seven sera bound only the IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033)chimera, indicating that binding required IA-2 specific amino acids within both 795-845 and 846-875, or that IA-2 residues 876-888 were important for binding. Mutation of glutamine 862 abolished binding in two of these sera, and mutation of residues 876, 877, 878, and 880 markedly reduced binding in two others. Six sera bound all three chimeras indicating that they contained multiple IA-2 specific PTP domain antibodies. In three of these sera, mutation of residues at positions 876, 877, 878, 880, and/or residues 862 and 822 reduced antibody binding by more than 50%. These findings indicate that glutamine at position 862, and residues 876-880 of the WPD loop of IA-2 are important for several of the IA-2 specific PTP domain epitopes.

Adolescent↗

Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: An objective of therapeutic intervention after cerebral ischemia is to promote improved functional outcome. Improved outcome may be associated with a reduction of the volume of cerebral infarction and the promotion of cerebral plasticity. In the developing brain, neuronal growth is concomitant with expression of particular proteins, including microtubule-associated protein 2 (MAP-2), growth-associated protein 43 (GAP-43), and cyclin D1. In the present study we measured the expression of select proteins associated with neurite damage and plasticity (MAP-2 and GAP-43) as well as cell cycle (cyclin D1) after induction of focal cerebral ischemia in the rat. METHODS: Brains from rats (n=28) subjected to 2 hours of middle cerebral artery occlusion and 6 hours, 12 hours, and 2, 7, 14, 21, and 28 days (n=4 per time point) of reperfusion and control sham-operated (n=3) and normal (n=2) rats were processed by immunohistochemistry with antibodies raised against MAP-2, GAP-43, and cyclin D1. Double staining of these proteins for cellular colocalization was also performed. RESULTS: Loss of immunoreactivity of both MAP-2 and GAP-43 was observed in most damaged neurons in the ischemic core. In contrast, MAP-2, GAP-43, and cyclin D1 were selectively increased in morphologically intact or altered neurons localized to the ischemic core at an early stage (eg, 6 hours) of reperfusion and in the boundary zone to the ischemic core (penumbra) during longer reperfusion times. CONCLUSIONS: The selective expressions of the neuronal structural proteins (MAP-2 in dendrites and GAP-43 in axons) and the cyclin D1 cell cycle protein in neurons observed in the boundary zone to the ischemic core are suggestive of compensatory and repair mechanisms in ischemia-damaged neurons after transient focal cerebral ischemia.

Animals↗

Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon.

The three major capsid proteins of adeno-associated virus type 2 (AAV2) virions are designated A, B, and C and have molecular sizes of 90, 72, and 60 kDa, respectively. These proteins are related, and genetic studies have shown they are encoded by a long open reading frame located in the right half of the genome. The coding capacity distal to the first ATG in this reading frame is only 503 amino acids (i.e., a protein about the size of protein C), but an open frame sequence devoid of ATG codons extends upstream for an additional 184 codons. Although the amino terminus of the C capsid protein is blocked, partial amino acid sequence analyses of peptides from C have confirmed that it is encoded within the portion of the reading frame distal to the first ATG at nucleotide (nt) location 2810. The amino terminus of the B capsid protein is not blocked, and its sequence begins with alanine. The triplet encoding this alanine lies 64 codons upstream from the initiation site for protein C and is immediately preceded by the threonine codon, ACG, at nt 2615. This ACG codon lies in the most favorable sequence context for protein synthesis initiation. All three AAV2 capsid proteins are labeled in vitro with formyl[35S]methionyl-tRNAf, indicating that synthesis of each protein is initiated independently. Our data suggest that the nt 2615 ACG codon directs the methionyl-tRNA-dependent initiation of the AAV2 B capsid protein. Proteins B and C may be synthesized from the same mRNA species and their relative abundance could be determined by the efficiencies of their respective initiation codons.

Amino Acid Sequence↗

Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state.

Acetylation↗

Hypoxia-induced IL-6 production is associated with activation of MAP kinase, HIF-1, and NF-kappaB on HEI-OC1 cells.

In the present study, we investigated the signal transduction pathways of expression of IL-6 in the desferrioxamine (DFX)-stimulated cochlear auditory cell line, HEI-OC1 cells. DFX increased the expression of HIF-1alpha and NF-kappaB in HEI-OC1 cells. DFX significantly increased the production of IL-6 (P<0.05) and expression of IL-6 mRNA but did not affect TNF-alpha production. DFX also induced the activation of mitogen-activated protein kinase (MAPK) including p38, ERK, and JNK on HEI-OC1. Increased IL-6 by DFX was significantly inhibited by p38 inhibitor, SB203580 (about 72% inhibition, P=0.027) but not ERK inhibitor, PD98059 or JNK inhibitor, SP600125. SB203580 inhibited the expression of IL-6 mRNA. Increased IL-6 production was partially inhibited by treatment of iron (HIF-1 inhibitor) or pyrriolidine-dithiocarbamate (PDTC, NF-kappaB inhibitor). DFX also induced IL-6 production and HIF-1alpha expression in the inner ear. We demonstrated the regulatory effects of MAPK, HIF-1alpha, and NF-kappaB on DFX-induced IL-6 production in a HEI-OC1 for the first time. In conclusion, these data indicate that regulation of inflammatory cytokine IL-6 by DFX, through mimicking hypoxic conditions, might explain its beneficial effect in the treatment of hypoxia-induced inner ear diseases.

Animals↗

Mapping a membrane-associated conformation of colicin Ia.

Channel-forming colicins exist in at least two different membrane-associated conformations: a voltage-independent closed-channel state and a voltage-dependent open-channel state. In a voltage-independent membrane-associated conformation, we find that two major regions of colicin Ia are protected from pepsin proteolysis after association with negatively charged membranes. In contrast, colicin Ia is rapidly and completely proteolyzed in the absence of membranes. The major protected region includes an electrophysiologically defined C-terminal channel-forming domain as well as 96 residues upstream of this region. Approximately 100 residues spanning Ala79- approximately Arg189 within the N-terminal domain are protected as well. The first N-terminal 76 residues of colicin Ia and a large region which includes much of the putative central receptor-binding domain are not protected from proteolysis. Both N- and C-termini of protected peptides have been identified using a combination of gel electrophoresis, N-terminal sequencing, and mass spectrometry, thereby defining specific residues that are located on the outside of the lipid bilayer. These data suggest a role for regions other than the electrophysiologically defined C-terminal channel-forming domain in membrane insertion and channel formation.

Amino Acid Sequence↗

Mapping conditioned taste aversion associations using c-Fos reveals a dynamic role for insular cortex.

Novel tastes are more effective than familiar tastes as conditioned stimuli (CSs) in taste aversion learning. Parallel to this, a novel CS-unconditioned stimulus (US) pairing induced stronger Fos-like immunoreactivity (FLI) in insular cortex (IC), amygdala, and brainstem than familiar CS-US pairing, suggesting a large circuit is recruited for acquisition. To better define the role of IC, the authors combined immunostaining with lesion or reversible inactivation of IC. Lesions abolished FLI increases to novel taste pairing in amygdala, suggesting a role in novelty detection. Reversible inactivation during taste preexposure increased FLI to familiar taste pairing in amygdala and brainstem. The difference between temporary inactivation, which blocked establishment of "safe" taste memory, and lesions points to a dual role for IC in taste learning.

Amygdala↗

Semantic and visual factors affecting the representation of map information.

In past research evidence has been found for both mental imagery and propositional hierarchies in subjects' map representations. How the visual and semantic factors associated with maps influence the use of one form of representation or another was the subject of the study reported here. Subjects were required to make relational judgments about city pairs. The results of experiment 1 indicated that superordinate relationships (the relationship of the counties of which the cities were members) affected only the most difficult perceptual judgments, but affected all judgments made from memory. Experiment 2 was done to determine the extent to which these findings were attributable to perceptual distortions rather than propositionally stored rules. Results of experiment 3 showed the degree of perceptual distinction necessary for propositional rules to have no significant influence on relational judgments.

Analysis of Variance↗

Familial osteoarthritis of the hip joint associated with acetabular dysplasia maps to chromosome 13q.

Genetic factors have been implicated in osteoarthritis (OA), particularly in OA of the hip joint (hip OA). Several instances of familial hip OA that show distinctive modes of inheritance but that differ from chondrodysplasia have been reported. Here, we report the characterization of a large Japanese family with an inherited disease of the hip that is indistinguishable from common hip OA, as evidenced by clinical symptoms and radiographs of the joint. This family contained eight patients in 4 generations. Affected individuals develop pain in the hip joint during adolescence, and the disease progresses to severe crippling before age 60 years. Patients generally are in good health, height is not reduced, and there is no extraskeletal involvement suggestive of chondrodysplasia. The skeletal change is bilateral acetabular dysplasia followed by OA, which occurs after age approximately 40 years and is indistinguishable from idiopathic nonfamilial dysplastic hip OA. This trait shows autosomal dominant inheritance, with a considerably consistent phenotype. Genomewide screening revealed linkage at chromosome 13q22, and haplotype analysis narrowed the locus to a 6.0-cM interval between markers D13S1296 and D13S162, with a maximal multipoint LOD score of 3.57. The family described here represents a novel genetic entity as a monogenic form of hip OA. Its further characterization can aid in elucidating the etiology and pathogenesis of a common idiopathic form of OA.

Chromosome Mapping↗

Expanded Chromatin Accessibility Mapping Explains Genetic Variation Associated with Complex Traits in Liver.

Genome-wide association studies (GWAS) have identified thousands of loci associated with a variety of common, complex human traits. Recent efforts have focused on characterizing chromatin accessibility to discover regulatory elements that modify the expression of nearby genes, suggesting that trait associations are mediated through changes in gene regulation. Genetic variants associated with differences in chromatin accessibility, known as chromatin accessibility quantitative trait loci (caQTLs), are established contributors to gene expression differences, providing mechanistic hypotheses for signals identified by GWAS. Using the assay for transposase-accessible chromatin with sequencing (ATAC-seq), we assessed chromatin accessibility in 189 diverse human liver samples, identifying over two million accessible chromatin regions enriched for gene regulatory features and, in 175 of these samples, over 14,000 caQTLs. Focusing subsequently on liver-relevant complex traits, we obtained publicly available blood lipids GWAS data and identified 157 loci where caQTLs, expression quantitative trait loci (eQTLs), and GWAS signals colocalized. This generated specific molecular hypotheses about regulatory elements, affected genes, and, in some cases, implicated transcription factors. Finally, we enumerated the set of blood lipid trait signals that lack an obvious proposed mechanism beyond catalogs of liver caQTLs and eQTLs. After integrating 10 multi-omic QTL regulatory mechanism datasets whilst considering limitations in statistical power, we found that approximately 20% of blood lipid GWAS signals lacked a statistical link to a proposed mechanism. Our results demonstrate the value of integrating multiple genomic datasets to improve understanding of GWAS signals, while emphasizing the need for additional experimental approaches to fully characterize complex trait associations.

Journal Article↗

Surface-based atlases of cerebellar cortex in the human, macaque, and mouse.

This study describes surface reconstructions and associated flat maps that represent the highly convoluted shape of cerebellar cortex in three species: human, macaque, and mouse. The reconstructions were based on high-resolution structural MRI data obtained from other laboratories. The surface areas determined for the fiducial reconstructions are about 600 cm(2) for the human, 60 cm(2) for the macaque, and 0.8 cm(2) for the mouse. As expected from the ribbon-like pattern of cerebellar folding, the cerebellar flat maps are elongated along the axis parallel to the midline. However, the degree of elongation varies markedly across species. The macaque flat map is many times longer than its mean width, whereas the mouse flat map is only slightly elongated and the human map is intermediate in its aspect ratio. These cerebellar atlases, along with associated software for visualization and for mapping experimental data onto the atlas, are freely available to the neuroscience community (see http:/brainmap.wustl.edu).

Animals↗

Insulin-dependent diabetes mellitus is associated with genes that map to the right of the class I RT1.A locus of the major histocompatibility complex of the rat.

Previous studies have demonstrated that the presence of at least one u-haplotype of the rat major histocompatibility complex (MHC), RT1, is a necessary but not sufficient condition for the development of overt diabetes mellitus. The present studies were undertaken to determine which portion of the RT1 gene complex is necessary for the occurrence of diabetes. We crossed hooded diabetic rats (RT1.AuBuDu) with PVGr8 rats (RT1.AaBuDu). F1 animals were mated to give 82 F2 animals and backcrossed with the hooded diabetics to produce 41 backcross animals. Diabetes occurred in animals with all three possible RT1.A genotypes. The diabetes was similar to that seen in BB rats and in hybrid strains developed from them. An immunoregulatory defect was marked by decreased percentage of peripheral blood lymphocytes staining with w3/25 monoclonal antibody, by an increased percentage of peripheral blood lymphocytes binding a mouse ascites control protein, and by decreased responsiveness of peripheral blood lymphocytes to stimulation by concanavalin A. We conclude that the u-allele of the class I A-locus gene product is not necessary for susceptibility to the development of diabetes in the rat. Therefore, either genes coding for the class II products of the u-haplotype or genes in linkage disequilibrium with these genes and mapping to the right of the A locus provide the permissive condition. Furthermore, the data suggest, but do not prove, that the u-haplotype derived from a strain remote from the BB rat can confer this susceptibility to the development of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification.

A cDNA encoding the novel drug resistance gene, LRP (originally termed lung resistance-related protein), was isolated from HT1080/DR4, a 220-fold doxorubicin-resistant human fibrosarcoma cell line which displays a multidrug resistance phenotype and overexpresses the multidrug resistance protein (MRP) but does not overexpress P-glycoprotein encoded by the MDR1 gene. Using the full-length 2.8-kb cDNA probe, the gene for LRP was regionally localized to the 16p13.1-16p11.2 chromosomal segment in human metaphases. Dual color fluorescence in situ hybridization studies refined the localization of LRP to 16p11.2, a location approximately 27 cM proximal to MRP (16p13.1). Two color hybridization studies indicated that HT1080/DR4 fibrosarcoma cells contain amplification of both the MRP and LRP genes in a striking striped pattern in the homogeneously staining region, hsr(7)(p12p15). In contrast, only amplified MRP gene sequences were contained within the homogeneously staining region, hsr(18q). Amplification of LRP was not identified in any of seven other drug-resistant tumor cell lines characterized by 20-300-fold levels of doxorubicin resistance, including two cell lines known to overexpress LRP (SW1573/2R120 and GLC4/ADR). Amplified MRP gene sequences were identified in H69AR, GLC4/ADR, and HL-60/AR whereas only MDR1 gene amplification was observed in the S1B120 colon carcinoma cell line. These data indicate that although both the MRP and LRP genes map to the short arm of chromosome 16, they are rarely coamplified and are not normally located within the same amplicon. A key role for chromosome breakage in gene amplification is supported by the presence of non-random karyotypic anomalies near the MRP and LRP normal cellular loci.

Chromosome Mapping↗