Combinatorial topography and cell-type specific regulation of the ERK pathway by dopaminergic agonists in the mouse striatum

Therapeutic agents and drugs of abuse regulate the extracellular signal-regulated kinase (ERK) cascade signaling in the medium-sized spiny neurons (MSNs) of the striatum. However, whether this regulation is associated with specific cortical and thalamic inputs has never been studied. We used Drd2-EGFP BAC-transgenic mice to undertake a topographical and cell-type specific analysis of ERK phosphorylation and two of its downstream targets histone H3 and ribosomal protein S6 (rS6) in the dorsal striatum following injection of SKF81297 (D1R-like agonist), quinpirole (D2R-like agonist) or apomorphine (non selective DA receptor agonist). In striatal areas receiving inputs from the cingulate/prelimbic, visual and auditory cortex, SKF81297 treatment increased phosphorylation of ERK, histone H3 and rS6 selectively in EGFP-negative MSNs of Drd2-EGFP mice. In contrast, no regulation was found in striatal region predominantly targeted by the sensorimotor and motor cortex. Apomorphine slightly enhanced ERK and rS6, but not histone H3 phosphorylation. This regulation occurred exclusively in EGFP-negative neurons mostly in striatal sectors receiving connections from the insular, visual and auditory cortex. Quinpirole administration inhibited basal ERK activation but did not change histone H3 and rS6 phosphorylation throughout the rostrocaudal axis of the dorsal striatum. This anatomo-functional study indicates that D1R and D2R agonists produce a unique topography and cell-type specific regulation of the ERK cascade signaling in the mouse striatum, and that those patterns are closely associated with particular cortical and thalamic inputs. This work evidences the need of a precise identification of the striatal areas under study to further understand striatal plasticity.     

Cytogenetic study of a papillary thyroid carcinoma with a rearranged chromosome 10.

Cytogenetic findings of a multifocal papillary thyroid cancer and a metastatic lymph node from a 29-year-old white female patient are reported. Two clonal aberrations were observed: a trisomy 7 in one nodule, and a rearranged chromosome 10 in a separate nodule and in a lymph node. The rearrangement of 10q described here is similar to other published cases and is relevant for interpreting the molecular findings associated with thyroid cancer.     

Gal80 intersectional regulation of cell-type specific expression in vertebrates

Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.     

Structural analysis of a rare rearranged Y chromosome and its bearing on genotype-phenotype correlation

We report on a 9-year-old boy with a rare rearranged Y chromosome and borderline short stature (-2.0 SD). Standard metaphase chromosome analysis indicated a 46,X,i(Y)(q1O) karyotype, but high resolution G-banding showed an asymmetric band pattern for the rearranged Y chromosome. FISH and DNA studies for a total of 15 different Y chromosomal loci or regions showed that the rearranged Y chromosome was accompanied by: 1) a partial deletion of the short arm pseudoautosomal region (PAR1) involving SHOX, with the breakpoint distal to DXYS85; and 2) a partial duplication of Yq, with the breakpoint proximal to DAZ. The karyotype was determined as 46,X,?i(Y)(q1O).ish der(Y)(Yqter--> Yp11.3::Yq11.2-->Yqter)(DAZ++,DYZ3+,SRY +, SHOX-). The X chromosome and the autosomes were normal. The results suggest that haploinsufficiency of SHOX is primarily responsible for the borderline short stature, and that the deletion of the PAR1 may result in spermatogenic failure due to defective X-Y pairing and recombination in the PAR1.     

Identification of a neocentromere in a rearranged Y chromosome with no detectable DYZ3 centromeric sequence

An 18-year-old woman was evaluated because of primary amenorrhea and hypogonadism. Chromosome analysis from peripheral blood lymphocytes revealed a nonmosaic 46,X,+mar constitution. The marker was shown to be a rearranged Y chromosome consisting of an inverted duplication of the long arm: rea(Y)(qter-q11::q11-qter). Deletion mapping analysis with Y-specific STS showed that the marker lacked Yp and Y-centromeric (DYZ3) sequences, but it was positive for Yq sequences tested. Fluorescence in situ hybridization analysis with Y and X chromosome centromeric and pancentromeric probes showed no hybridization signals. The marker chromosome is present in 100% of the cells; therefore, it is mitotically stable despite the absence of DYZ3 centromeric sequence. Hybridization with CENP-A and CENP-C specific antibodies localized a neocentromere close to the breakpoint.     

Assignment of the lactotransferrin gene to human chromosome 3 and to mouse chromosome 9

Lactotransferrin (LTF), a member of the transferrin family of genes, is the major iron-binding protein in milk and body secretions. The amino acid sequence of LTF consists of two homologous domains homologous to proteins in the transferrin family. Recent isolation of cDNA encoding mouse LTF has expedited the mapping of both mouse and human LTF genes. Southern blot analysis of DNA from mouse-Chinese hamster and human-mouse somatic cell hybrids maps the LTF gene to mouse chromosome 9 and to human chromosome 3, respectively. Furthermore, analysis of cell hybrids containing defined segments of human chromosome 3 demonstrates that the gene is located in the 3q21-qter region. These results suggest that LTF and associated genes of the transferrin family have existed together on the same chromosomal region for 300–500 million years.     

Assignment of three human markers in chromosome 21q11 to mouse chromosome 16

Three unique sequence microclones from human chromosome region 21q11 were assigned to mouse chromosome 16 using a mouse/Chinese hamster cell hybrid 96Az2 containing a single mouse chromosome 16. This comparative mapping provides further homology between human chromosome 21 and mouse chromosome 16 to include the very proximal portion of the long arm of human chromosome 21. Since this part of human chromosome 21 is associated with mental retardation in Down syndrome individuals, its homologous mouse region should also be included in the construction of mouse models for studying Down syndrome phenotypes including mental retardation.     

Second meiotic nondisjunction of the rearranged chromosome in a familial reciprocal 5/13 translocation

We describe a 20-week-gestation male fetus with partial dup(5p) and proximal dup(13q), 47,XY,t(5;13)(p15;q21), + der(13)t(5;13)(p15;q21) mat. This finding is attributable to second meiotic nondisjunction of the rearranged chromosome in a maternal balanced reciprocal translocation. To the best of our knowledge, there have been only 3 previous reports of a similar error in the segregation of the rearranged chromosomes. For the first time evidence has been given that this unusual segregation is due to maternal second meiotic nondisjunction, using QFQ banding heteromorphisms. Second meiotic malsegregation should be taken into account in the consideration of reproductive problems in carriers of balanced translocations.     

Partial monosomy or trisomy resulting from crossing over within a rearranged chromosome 1

Two sibs, both monosomic for the same portion of lq (q25q32), had similar severe mental and physical retardation. An older sib was found to be trisomic for the same portion of 1q and less severely affected. These partially monosomic and partially trisomic No. 1 chromosomes resulted from meiotic crossover in the mother, who is a balanced hetero-zygote of the type 46.XX, ins (1) (p32q25q32).     

Regional and cell-type specific distribution of HDAC2 in the adult mouse brain

The effects of epigenetics on brain functions are not completely understood, but histone deacetylases (HDACs) are known to affect brain function and dysfunction by mediating the acetylation status of target proteins, thereby affecting gene expression. The current study used immunochemistry to illuminate the regional distribution of one member of the HDAC family, HDAC2, in the C57BL/6J mouse brain. Our data show that HDAC2 is ubiquitously expressed throughout the mouse brain and is localized primarily within the cell nucleus. Using double-immunofluorescence, we demonstrated HDAC2 expression in neuronal cells, including cholinergic, serotonergic and catecholaminergic neurons, as well as postsynaptic glutamatergic and GABAergic neurons. HDAC2 was also observed in oligodendrocytes, but not in astrocytes or microglia. These detailed immunological studies illuminate the distribution of HDAC2 throughout the mouse brain and will facilitate investigation of the roles of HDAC2 in brain function and neurological disorders.     

A new region of synteny between human chromosome 1p22 and mouse chromosome 5

By constructing a physical map of the p22 region of human chromosome 1 we have been able to show the relative orientation of four genes; GFI1, NB4S/EVI5, RPL5 and DR1. Analysis of the mouse physical map shows that the murine orthologs of these genes are located on mouse chromosome 5. Through this analysis we have established a new region of synteny between mouse chromosome 5 and human chromosome region 1p22.     

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21qHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21qHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21qHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21qHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21qHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21qHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.     

Development of a human herpesvirus 6 species-specific immunoblotting assay

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.     

Location and sequence of rearranged immunoglobulin genes in human thymus

Thymic B cells are a proliferating B cell population concentrated in normal thymic medulla. They are large cells, some with dendritic morphology, and are not associated with any organized follicular structure. Previous work in this laboratory has shown that most of these B cells are surrounded by tightly adherent thymocytes. The literature on human thymic B cells contains many inconsistencies. There is no consensus on whether they express CD5. Even the existence of thymic B cells has been questioned. In this study we have undertaken the first analysis of rearranged immunoglobulin (Ig) genes, looking in particular for evidence of affinity maturation. The Ig V_H genes of human thymic B cells in this study are those of the fetal repertoire, though the resemblence to fetal Ig genes is limited in other respects. They are mostly unmutated, but the presence of mutated sequences suggests that this is not a uniform population, as has been previously indicated by phenotypic studies.     

Construction of a physical map on mouse and human chromosome 1: comparison of 13 Mb of mouse and 11 Mb of human DNA.

Long range restriction site maps of 13 Mb of mouse chromosome 1 and 11 Mb of human chromosome 1 were constructed using a framework provided by a detailed mouse genetic map. Where an unambiguous gene order could be determined in both species (14 genes), the human and mouse orders were identical. In addition, the distances between markers in the mouse and human were similar except for one region of the conserved linkage group where we could detect a larger distance in the mouse compared to the human. These data support the use of comparative mapping in physical map construction and further suggest the value of using mouse genetics to help define human disease loci.     

Structural analysis of a rare rearranged Y chromosome and its bearing on genotype–phenotype correlation

We report on a 9‐year‐old boy with a rare rearranged Y chromosome and borderline short stature (−2.0 SD). Standard metaphase chromosome analysis indicated a 46,X,i(Y)(q10) karyotype, but high resolution G‐banding showed an asymmetric band pattern for the rearranged Y chromosome. FISH and DNA studies for a total of 15 different Y chromosomal loci or regions showed that the rearranged Y chromosome was accompanied by: 1) a partial deletion of the short arm pseudoautosomal region (PAR1) involving SHOX, with the breakpoint distal to DXYS85; and 2) a partial duplication of Yq, with the breakpoint proximal to DAZ. The karyotype was determined as 46,X,?i(Y)(q10).ish der(Y)(Yqter→ Yp11.3::Yq11.2→Yqter)(DAZ++,DYZ3+,SRY+, SHOX−). The X chromosome and the autosomes were normal. The results suggest that haploinsufficiency of SHOX is primarily responsible for the borderline short stature, and that the deletion of the PAR1 may result in spermatogenic failure due to defective X‐Y pairing and recombination in the PAR1. Am. J. Med. Genet. 92:256–259, 2000. © 2000 Wiley‐Liss, Inc.     

几种人源乳酸杆菌的免疫调节及抑瘤作用

目的探讨人阴道内分离出的3株乳酸杆菌对巨噬细胞的调节和拮抗小鼠宫颈癌U14的作用。方法建立KM小鼠U14移植瘤动物模型,随机分组,以模型组、顺铂组(DDP)分别作为阴性、阳性对照组。乳酸杆菌组小鼠腹腔注射乳酸杆菌1010个/只,连续10 d;测定小鼠腹腔巨噬细胞产生TNF-α、NO的水平和肿瘤抑制率。结果与模型组比较,3株乳酸杆菌处理组的小鼠腹腔巨噬细胞产生TNF-α、NO的水平明显增高,均能抑制KM小鼠U14移植瘤生长。结论乳酸杆菌具有抑制小鼠宫颈癌的作用,其抑瘤机制可能与激活巨噬细胞产生TNF-α和NO有关。     

The short arm of chromosome 6 is nonrandomly rearranged in secondary myelodysplastic syndromes

Eight patients with a myelodysplastic syndrome showed a structural rearrangement of the short arm of chromosome #6 involving the distal segment 6p22----6pter. In four cases the myelodysplastic disorder appeared after treatment with chemo- and/or radiotherapy. Cytogenetically, the 6p anomaly was consistently associated with abnormalities of chromosome #5 and/or #7 in seven of eight cases. We believe we identified a new site on 6p that is nonrandomly involved in iatrogenically and possibly also environmentally induced malignant hematologic disorders.     

Over-representation of species-specific vocalizations in the awake mouse inferior colliculus

Social vocalizations are particularly important stimuli in an animal's auditory environment. Because of their importance, vocalizations should be strongly represented in auditory pathways. Mice commonly emit ultrasonic vocalizations with spectral content between 45 and 100 kHz. However, there is limited representation of these ultra-high frequencies (particularly those greater than 60 kHz) throughout the ascending auditory system. Here, we show that neurons in the inferior colliculus (IC) of mice respond strongly to conspecific vocalizations even though the energy in the vocalizations is above the neurons' frequency tuning curves. This results in an over-representation of species-specific vocalizations in the IC. In addition, neurons in mouse IC show selectivity among different vocalizations. Many vocalization-responsive neurons do not respond to the individual ultrasonic frequencies contained within the vocalizations, but they do respond to combinations of ultrasonic tones if the difference between the tones is within the excitatory frequency tuning curve. The combinations of tones that elicit responses are the quadratic and/or cubic intermodulation distortion components that are generated by the cochlea. Thus, the intermodulation distortions in the cochlea may provide a previously overlooked mechanism for auditory processing of complex stimuli such as vocalizations. The implication of these findings is that nonlinear interactions of frequencies, possibly caused by distortions in the system, may be used to enhance the sensitivity to behaviorally important stimuli.