Adaptative decrease in expression of the mRNA for uncoupling protein and subunit II of cytochrome c oxidase in rat brown adipose tissue during pregnancy and lactation.

Uncoupling-protein (UCP) mRNA expression is decreased to 15% of virgin control levels between days 10 and 15 of pregnancy, and remains at these low values during late pregnancy and lactation. Abrupt weaning of mid-lactating rats causes a slight but significant increase in UCP mRNA. Expression of mRNA for subunit II of cytochrome c oxidase (COII) decreased to half that of virgin control in late pregnancy and during lactation. Whereas COII mRNA expression is in step with the known modifications of brown-fat mitochondria content during the breeding cycle of the rat, UCP mRNA expression appears to be diminished much earlier than the mitochondrial proton-conductance-pathway activity. On the other hand, the reactivity of brown fat to increase expression of UCP and COII mRNAs in response to acute cold or noradrenaline treatment is not impaired during lactation.     

Splenomegaly induced by recombinant human granulocyte-colony stimulating factor in rats

Spontaneous ruptures of the spleen have been observed in donors and patients with malignancy during mobilization of peripheral blood stem cells. Thus, we investigated the morphological and histological alteration of the spleen, and mRNA expression levels and activities of the DNA-synthesizing enzymes thymidylate synthase (TS) and thymidine kinase (TK) in the splenic cells, of rats treated with recombinant human granulocyte-colony stimulating factor (rhG-CSF). Daily injections of rhG-CSF for 5 or 7 days slightly enhanced the splenic weight. Single or 3-day treatment with rhG-CSF markedly enhanced the number of granulocytes in peripheral blood. Expression levels of TS and TK mRNA in the splenic cells were significantly increased 6 hours after rhG-CSF treatment. Early expression of TS and TK mRNA in the splenic cells may indicate a reseeding of hematopoietic cells from the bone marrow. Daily injections with rhG-CSF enhanced the TS and TK activities in the splenic cells. As continuous elevations of DNA-synthesizing enzyme activity and spleno-megaly are suggestive of a possible splenic rupture, the monitoring of peripheral granulocytes and splenic size is necessary during the rhG-CSF treatment.     

Murine cytomegalovirus downregulates interleukin-17 in mice with retrovirus-induced immunosuppression that are susceptible to experimental cytomegalovirus retinitis

Interleukin-17 (IL-17), a pro-inflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by splenic CD4+ T cells. Based on additional studies using IL-10 ?/? mice infected systemically with MCMV and IL-10 ?/? mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10.     

Ontogeny and perinatal modulation of gene expression in rat brown adipose tissue. Unaltered iodothyronine 5'-deiodinase activity is necessary for the response to environmental temperature at birth

We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit II of cytochrome-c oxidase (COII) and lipoprotein lipase in brown adipose tissue during the fetal and postnatal periods. Moreover, we have determined whether these parameters can be modulated by ambient temperature in the early hours after birth, and at which point in development this sensitivity first appears. UCP gene expression in the fetal and neonatal period has particular features when compared with overall mitochondriogenesis (COII mRNA expression) or with the expression of lipoprotein lipase mRNA. There is a specific induction of UCP gene expression between days 18 and 19 of pregnancy followed by a specific increase of UCP gene expression in utero and a further increase after birth. The acquisition of the physiological apparatus capable of the response to UCP and lipoprotein lipase gene expression to the environmental temperature is not achieved until the last day of fetal development. This result suggests that mechanisms of beta-adrenergic modulation of gene expression in brown fat are already established at birth. From an experiment on iopanoic acid treatment of pregnant mothers, it was concluded that iodothyronine 5'-deiodinase activity is not necessary for the expression of the mRNAs for UCP, COII and lipoprotein lipase in the fetus whereas it is necessary for the acquisition of temperature sensitivity to these parameters at birth.     

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.     

Downregulation of CXCL12 signaling and altered hematopoietic stem and progenitor cell trafficking in a murine model of acute Anaplasma phagocytophilum infection

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4-8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2-6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.     

Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells.

The progressive differentiation of both normal rat osteoblasts and HL-60 promyelocytic leukemia cells involves the sequential expression of specific genes encoding proteins that are characteristic of their respective developing cellular phenotypes. In addition to the selective expression of various phenotype marker genes, several members of the heat shock gene family exhibit differential expression throughout the developmental sequence of these two cell types. As determined by steady state mRNA levels, in both osteoblasts and HL-60 cells expression of hsp27, hsp60, hsp70, hsp89 alpha, and hsp89 beta may be associated with the modifications in gene expression and cellular architecture that occur during differentiation. In both differentiation systems, the expression of hsp27 mRNA shows a 2.5-fold increase with the down-regulation of proliferation while hsp60 mRNA levels are maximal during active proliferation and subsequently decline post-proliferatively. mRNA expression of two members of the hsp90 family decreases with the shutdown of proliferation, with a parallel relationship between hsp89 alpha mRNA levels and proliferation in osteoblasts and a delay in down-regulation of hsp89 alpha mRNA levels in HL-60 cells and of hsp89 beta mRNA in both systems. Hsp70 mRNA rapidly increases, almost twofold, as proliferation decreases in HL-60 cells but during osteoblast growth and differentiation was only minimally detectable and showed no significant changes. Although the presence of the various hsp mRNA species is maintained at some level throughout the developmental sequence of both osteoblasts and HL-60 cells, changes in the extent to which the heat shock genes are expressed occur primarily in association with the decline of proliferative activity. The observed differences in patterns of expression for the various heat shock genes are consistent with involvement in mediating a series of regulatory events functionally related to the control of both cell growth and differentiation.     

Owl monkeys (Aotus) are highly divergent in mitochondrial Cytochromec Oxidase (COII) sequences

The evolutionary history and differentiation of the owl or night monkeys (Aotus),remain poorly resolved. Variation in pelage and skeletal morphology is relatively minor across their broad range, but cytogenetic studies have revealed that at least 12 karyotypically distinct forms exist, with 2Nchromosome numbers ranging from 46 to 58. We obtained DNA samples from three putative species- A. lemurinus, A. nancymae,and A. azarae-and five karyotypes (I, II, III, IV and VI), amplified the mitochondrial cytochrome coxidase subunit II gene (COII) via PCR and sequenced it. Comparisons of the sequences indicate relatively high levels of sequence divergence among the putative species (4.3 to 6.3%), and suggest that the taxa are genetically quite distinct and have likely experienced extended periods of isolated evolution. The levels of COII divergence represent approximately one- third of the levels found between divergent platyrrhine genera, such as Aotus, Saimiriand Callimico.Using estimates of substitution rates of COII evolution in hominoid primates,the estimated date of divergence for the AotusCOII sequences is 3.6 million years. The AotusCOII data support the existence of multiple species of Aotus,with origins predating late Pleistocene climatic events. Although A. nancymaeand A. azaraeboth live south of the Amazon River and have been considered members of the same species group, phylogenetic analysis of the COII sequences does not support a close relationship between them.     

Enhancement of c-erbA proto-oncogene expression by glucocorticoid hormones in S49.1 lymphoma cells

The modifications of the mRNA levels of the c-myc and c-erbA proto-oncogenes during the dexamethasone-induced decrease of S49.1 cell proliferation have been studied. The levels of c-myc mRNA decreased significantly between 3 and 18 h after dexamethasone (1 microM) treatment. In contrast, a significant increase in the levels of a 2.6 kb c-erbA mRNA was observed between 6 and 18 h after hormone treatment. Cycloheximide treatment of S49.1 cells increased the levels of c-erbA RNA and overcome the enhancing effect of dexamethasone on the expression of this proto-oncogene, suggesting that ongoing protein synthesis is necessary to elicit this hormone effect. The associated decrease of cell proliferation and changes in c-myc and c-erbA mRNA levels after dexamethasone treatment suggest that such oncogenes might be involved in the dexamethasone-mediated control of lymphoid cell growth.     

CD28 costimulation induces delta opioid receptor expression during anti-CD3 activation of T cells

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the delta-type opioid receptor (delta OR), but stimulation of mouse splenocytes with Con A results in induction of delta OR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit delta OR expression. However, delta OR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The delta OR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of delta OR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for delta OR expression because delta OR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.     

Effects of long-term estrogen treatment on IFN-gamma, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice.

Estrogens have been shown to markedly modulate the immune system. One mechanism by which estrogens could modulate the immune system is by regulating cytokines, an aspect not well-studied thus far. To address this issue, normal C57BL/6 orchiectomized mice were given estrogen and its effects on selected cytokines, interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and IL-4 in lymphocytes from a developmental organ (thymus) and a mature lymphoid organ (spleen) examined. Estrogen significantly increased IFN-gamma and IL-2 mRNA in concanavalin-A (Con-A) activated thymocytes, splenic lymphocytes, and in enriched splenic T cells. Estrogen had no marked effect on IL-4 mRNA. While estrogen increased IFN-gamma mRNA in Con-A activated unseparated splenic lymphocytes and enriched splenic T cells, a numerical increase in IFN-gamma was noticed only in the supernatants of Con-A activated unseparated splenic lymphocytes, but not in enriched splenic T cells. This suggests that for optimal secretion of IFN-gamma in estrogen-treated mice, co-stimulatory signals from antigen presenting cells are needed. Gender differences in IFN-gamma and IL-2 mRNA were also evident. Con-A activated splenic lymphocytes from gonadal-intact, untreated female had a pattern of numerical increase in IFN-gamma mRNA, and IFN-gamma and IL-2 protein levels compared to their male counterparts. Taken together, our data suggests that estrogens regulate the expression of cytokines, which could account in part, for the gender differences in immune capabilities.     

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-myc and resultant depression of proliferation. © 1996 Wiley-Liss, Inc.     

Genetic variation and population structure of the mosquito Anopheles jeyporiensis in southern China

Genetic differentiation among populations of Anopheles jeyporiensis was examined using 76 mtDNA COII sequences from 16 sites throughout southern China and northern Vietnam. The COII sequences are AT-rich (74.58%) and reveal high levels of diversity with 39 of 685 sites polymorphic and 50 different haplotypes present. Genetic variation is high within populations and significant geographical structure was detected at both population and regional levels. In the larger samples, the distributions of haplotypes suggest recent population expansion.