Down syndrome and the trisomy 16 mouse: impact of gene imbalance on brain development and aging.
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Aging↗
Biomedical subjects
R Reeves
Publications and source records attributed to R Reeves.
Influence of short term dietary cholesterol and fat on human plasma Lp[a] and LDL levels.
The relationship between plasma levels of Lp[a] and LDL was examined using dietary regimens. In 81 normolipidemic male outpatients, dietary cholesterol was increased by consuming six eggs per day from a mean (SD) level of 311 (162) to 1430 (198) mg per day. Mean (SD) LDL-cholesterol levels increased from 102 (26) mg/dl to 120 (33) mg/dl (P less than 0.001), while mean (SD) Lp[a] levels were 5.5 (6.1) mg/dl on the basal diet and 5.6 (6.4) mg/dl on the cholesterol-rich diet. No significant correlation was observed between increases in either LDL-cholesterol or apolipoprotein B to Lp[a], nor was there any relationship between individual baseline levels of Lp[a] and dietary-induced changes of Lp[a]. Fourteen of the 81 participants were reexamined under strict nutritional control. Four diets with 40% of calories as fat, but differing in the type of fat and the amount of cholesterol, were administered sequentially to all subjects. As expected, mean (SD) LDL-cholesterol and apolipoprotein B levels were highest on the saturated fat, high cholesterol diet (112 (32) mg/dl and 79 (22) mg/dl) and lowest on the polyunsaturated fat, low cholesterol diet (77 (27) mg/dl and 53 (18) mg/dl). In contrast, mean Lp[a] levels did not significantly change among the four diets (range 4.2-4.9 mg/dl). No correlation of Lp[a] responses with changes in plasma lipids, apolipoproteins, or lipoproteins was observed on any diet. These data suggest that determinants of plasma Lp[a] levels are distinctly different from the determinants of plasma LDL levels in normolipidemic males.
Adult↗
Primary structure of the putative human oncogene, pim-1.
A full-length (6.1-kb) human genomic pim-1 gene, together with its immediate 5'-upstream promoter sequence (Ppim) was isolated and sequenced. The human pim-1 gene shares an overall nucleotide (nt) sequence identity of 53% with the previously reported murine pim-1 gene. It consists of six exons and five introns and contains a protein-coding region that is identical in nt sequence to a full-length human pim-1 cDNA. The gene codes for a predicted Pim-1 protein of 313 amino acids (aa) with an Mr of 35,690 and a pI of 5.7. The deduced aa sequence of the human Pim-1 has 94% identity with the murine Pim-1 whereas the nt sequences of the two genes are 88% identical. All of the conserved aa residues of the mouse pim-1 gene, which are homologous to known protein kinases are conserved in the predicted human protein. The human Ppim region is very G + C-rich (69%) and shares greater than 80% identity with the murine Ppim. The Ppim has no TATA- or CAAT-box sequences but does contain a number of nt sequences similar to the putative binding sites of several presumptive transcription factors.
Amino Acid Sequence↗
The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure.
We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.
Adenine↗
Developmental expression of somatostatin in mouse brain. II. In situ hybridization.
The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.
Aging↗
IL-2 can enhance the cyclosporin A-mediated inhibition of Theileria parva-infected T cell proliferation.
The effect of cyclosporin A on the continuous proliferation of Theileria parva-infected T cells was tested and compared with its effect on the Con A-induced proliferation of bovine lymph node cells. The effect of rIL-2 on cyclosporin A-treated cells was also tested. Whereas the Con A-induced proliferation of bovine lymph node cells was completely inhibited by cyclosporin A, the continuous growth of T. parva-infected cells was only partly inhibited. In both cases the inhibition was accompanied by a reduction in the level of IL-2R/Tac mRNA and surface IL-2R expression. The cyclosporin A-mediated inhibition of Con-A stimulated lymphoblasts was, over a period of 5 days, largely abrogated by human rIL-2. In the short term, rIL-2 could also alleviate the growth inhibition of T. parva-infected cells caused by treatment with cyclosporin A. In the long term, however, rIL-2 enhanced the cyclosporin A-mediated inhibition of T. parva-infected cells, gradually leading to their complete growth arrest. This enhanced inhibition was accompanied by a further reduction in surface IL-2R expression, but not by a further decrease in the levels of steady state IL-2R/Tac mRNA. The fact that IL-2 can enhance the inhibition caused by cyclosporin A could be of relevance for the immunosuppressive activity of cyclosporin A.
Animals↗
Expression of mRNAs encoding mammalian chromosomal proteins HMG-I and HMG-Y during cellular proliferation.
The high mobility group chromosomal proteins HMG-I and HMG-Y are closely related isoforms that are expressed at high levels in rapidly dividing, undifferentiated mammalian cells. We analyzed HMG-I/Y mRNA levels at various cell cycle stages in murine NIH/3T3 fibroblasts partially synchronized by seeding from quiescent, contact-inhibited cultures. Flow microfluorometric analysis of DNA content demonstrated a comparable degree of synchronization in such seeded NIH/3T3 cell populations as is obtained by serum deprivation or other means and has the added advantage of avoiding the use of possibly detrimental inhibitors or metabolic starvation to induce such synchrony. We show that HMG-I/Y mRNA levels gradually increase in NIH/3T3 cells during the first 16 h after seeding (G0/G1 to late S phase), but thereafter remain constant, in contrast to the cell cycle-regulated expression of the histone H3 gene. Although there is a 6-fold increase in HMG-I/Y expression during the transition from quiescent to proliferating NIH/3T3 cells, there is a much greater difference in expression (15- to 50-fold) among different cell types, possibly related to their state of differentiation. The HMG-I/Y mRNAs appear to be very stable; there was no decrease in their levels 6 h after actinomycin D transcription termination. The proportion of HMG-I to HMG-Y mRNAs was greater in the human than in the murine cells examined, appeared to be greater in proliferating than in quiescent cells, and did not always correspond with the HMG-I to HMG-Y protein ratio.
Animals↗
Mechanisms regulating transient expression of mammalian cytokine genes and cellular oncogenes.
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Animals↗
Nucleotide sequence of human mitochondrial cytochrome c oxidase II cDNA.
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Amino Acid Sequence↗
Expression and characterization of the interleukin 2 receptor in Theileria parva-infected bovine lymphocytes.
We have previously shown that interleukin 2 receptors (IL2R) are constitutively expressed on the surface of bovine lymphocytes infected with the parasite Theileria parva (Dobbelaere, D.A.E. et al., Proc. Natl. Acad. Sci. USA 1988. 85: 4730). In the present work we characterized these further and showed that IL2R (Tac antigen) gene expression depended on the continuous presence of the parasite in the host cell cytoplasm. By Northern blot analysis we showed that elimination of the parasite, using a specific theilericidal drug, led to the arrest of Tac antigen mRNA expression. We also investigated receptor internalization and reappearance after receptor-mediated endocytosis. Binding of human recombinant interleukin 2 (hrIL2) to the bovine IL2R caused rapid internalization of the surface IL2/IL2R complex. Approximately 50% of 125I-labeled hrIL2 was internalized within 10 min. The reappearance of surface IL2R after ligand-mediated endocytosis was also studied. Fifty percent of the maximum level of free IL2R reappeared within 1-1.5 h, but approximately 12 h were needed to restore normal levels of free IL2R expression after blocking with excess unlabeled hrIL2.
Animals↗
Effect of dexamethasone on the expression of interleukin-2 in a mouse T cell line.
1. The effect of the synthetic glucocorticoid, dexamethasone (dex), on the production of interleukin-2 (IL-2) and IL-2 mRNA was examined in the mouse T cell line, LBRM-33.4A2. 2. Treatment of Concanavalin A (Con A)-stimulated LBRM-33 cells with low concentrations of dex (10nM) inhibited the production of IL-2 activity by approx. 70%, with a corresponding decrease in IL-2 mRNA levels. 3. In contrast, much higher concentrations of dex (up to 10 microM) inhibited the level of another Con A-inducible mRNA, c-myc mRNA, by only 30%, and did not affect beta-tubulin mRNA levels at all. 4. Thus it appears that inhibition by dex in stimulated LBRM-33 cells is specific for the expression of IL-2 mRNA. 5. Experiments with actinomycin D suggest that dex does not mediate its suppression of IL-2 mRNA accumulation by decreasing the stability of this message; rather, it appears that dex inhibits transcription of the IL-2 gene.
Animals↗
High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.
Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.
Animals↗
Persistent infection of rabbits with bovine leukemia virus associated with development of immune dysfunction.
Bovine leukemia virus (BLV) infection of rabbits provides a safe and relatively inexpensive in vivo mammalian system for the study of the mechanisms controlling expression of a unique group of lymphotropic retroviruses. This group of viruses, which includes C-type human T-lymphotropic virus types I and II and lentiviruslike human immunodeficiency virus type 1, possesses genes coding for "trans-activating" products. Rabbits experimentally inoculated with BLV became persistently infected, as demonstrated by a number of tests. All BLV-inoculated rabbits developed persistent serum antibody to BLV. Furthermore, all BLV-inoculated rabbits had peripheral blood mononuclear cells which, when stimulated, expressed the virus, as demonstrated by viral induction of syncytium formation in a BLV-susceptible fibroblast line. The presence of BLV in circulating cells was confirmed by using peripheral blood mononuclear cells from randomly selected BLV-inoculated rabbits, which showed the presence of viral reverse transcriptase activity, BLV transcriptional activity, or BLV proviral DNA. Additional tests showed that infected lymphocytes maintained in culture with recombinant human interleukin-2 formed multinucleated giant cells and produced virus when incubated in cytokine-containing medium. BLV-infected rabbits also showed alterations in several parameters associated with immunity, beginning 6 months after inoculation. Thirty-eight percent of infected rabbits developed abnormally low T-cell responses, as measured by phytolectin stimulation, and T-cell responses cycled between normal and abnormally low over a period of 20 to 24 months. Forty-four percent of rabbits infected for longer than 12 months suffered from recurrent conjunctivitis and rhinitis. By 24 months postinoculation, 28% of infected rabbits were dead or were killed because of poor clinical condition.
Animals↗
Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.
The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.
Animals↗
Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y.
The high-mobility-group protein HMG-I is a well-characterized nonhistone chromosomal protein that is preferentially expressed in rapidly dividing cells, binds to A. T-rich regions of DNA in vitro, and has been localized to particular regions of mammalian metaphase chromosomes. We isolated eight cDNA clones encoding HMG-I and its isoform HMG-Y from a human Raji cell cDNA library and detected blocks of nucleotide sequence rearrangements in the 5'-untranslated regions of these clones. In addition to this leader sequence variation, five of the eight cDNA clones had either a 33- or 36-base-pair in-frame deletion in their open reading frame (ORF); we found that this shortened ORF encodes the HMG-Y protein isoform. We present evidence that the 5'-untranslated-region and ORF heterogeneity of the cDNA clones is the result of alternative processing of RNA transcripts from a single functional gene. Several additional but probably nonfunctional HMG-I or HMG-Y gene copies exist in the human genome; we isolated and partially sequenced one of these pseudogenes and found that it is a processed HMG-Y retropseudogene.
Amino Acid Sequence↗
Characterization of the bovine receptor(s) for interleukin-2.
The binding properties of the bovine receptors (IL-2R) for the lymphokine interleukin-2 (IL-2) have been examined using activated bovine lymphoid cells and human recombinant [125I] IL-2. The results of these binding studies indicate that the bovine IL-2R in many ways resembles the receptors for human and mouse IL-2, but with some differences. Equilibrium binding experiments revealed the presence of two classes of bovine IL-2R, one with a KD approximately 20 pM, representing approximately 400-1300 sites per cell, the other with a KD approximately 6 nM, representing approximately 20,000-50,000 sites per cell. A study of the time course of IL-2R appearance on the cell surface of activated bovine lymphocytes showed that both high- and low-affinity receptors appear rapidly following stimulation, with maximum levels of expression being reached within about 48-96 hr. Lymphoid cell proliferation, as monitored by [3H]thymidine [( 3H]TdR) incorporation, increased in parallel with the expression of high-affinity IL-2R. Measurements of the association/dissociation kinetics showed that IL-2 binds to (t1/2 approximately 10 seconds), and dissociates from (t1/2 approximately 20 seconds) the low-affinity bovine IL-2R very rapidly. In contrast, IL-2 binds rapidly to (t1/2 approximately 40 seconds), but dissociates slowly from (t1/2 approximately 8.5 hr) the high-affinity bovine IL-2R. In previous work, our laboratory has molecularly cloned the cDNA coding for the bovine IL-2 and IL-2R (p55, Tac) proteins. Comparisons of the deduced amino acid sequences of these bovine proteins with those of the homologous human and mouse proteins revealed a high degree of evolutionary conservation among these mammalian proteins. Our present IL-2/IL-2R binding studies are also consistent with such a close evolutionary relationship, but leave unresolved the molecular basis for the previously observed species specificity of the bovine IL-2/IL-2R receptor-ligand system.
Animals↗
Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y).
A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.
Amino Acid Sequence↗
Evidence for two discrete phases of IL-2 production in bovine lymphocytes.
Regulation of IL-2 production in Con A-stimulated bovine lymph node cells was studied by following the time course of IL-2 synthesis and secretion over a 24-h period. A biphasic time course curve of IL-2 secretion was observed after stimulation with Con A alone or with Con A plus PMA. Both phases of IL-2 production were confirmed by three separate biochemical techniques: IL-2 protein bioassay, mRNA Northern blots, and anti-IL-2 antibody detection. Bovine IL-2 transcripts were detected as early as 2 h after Con A stimulation. The early phase of IL-2 protein secretion was initially detected 3 h after Con A stimulation, and the late phase occurred between 10 and 16 h. Both phases of IL-2 production were enhanced when PMA was added to the Con A stimulation. Each phase of IL-2 protein secretion was preceded by the accumulation of IL-2 mRNA.
Animals↗