Trmt112 gene expression in mouse embryonic development

Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.     

Regulation of cytokine gene expression by adjuvants in vivo

Antibody isotype affects biological activity of the antibodies and therefore should be considered in prevention of disease by vaccination. In previous reports, we demonstrated that adjuvants affect the antibody isotype switching process and favour the production of certain isotypes. The present study extends these findings and shows fundamental differences in the cytokine induction pattern according to the adjuvant used. Cytokine mRNA levels were determined by in situ RNA–RNA hybridization performed on splenocytes isolated from mice injected with different adjuvants. The results revealed that Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Al(OH)₃ and QuilA administration results in a type-2 (humoral) response, increasing IL-4, IL-5 and IL-13 gene expression, while poly I:C exhibits a type-1 (cell-mediated) response, increasing the production of interferon-gamma (IFN-γ), IL-2 and IL-6 mRNA. Finally, BeSO₄ and poly A:U augment IL-5 and IL-6 mRNA production, while lipopolysaccharide (LPS) and LiCl augment IL-6 and tumour necrosis factor-alpha (TNF-α) mRNA production. Also, the adjuvants appear capable of overcoming the inherent IL-2/IFN-γ and IL-4 dichotomy of C57Bl/6 and BALB/c mice, respectively, in response to cellular antigens such as Leishmania and herpessimplex virus (HSV). The overall data suggest that adjuvants direct the isotype switching process via induction of certain cytokines, a finding that can be useful in selection of the most efficient isotype of protective antibodies for disease prevention by vaccination.     

MeCP2 expression and promoter methylation of cyclin D1 gene are associated with cyclin D1 expression in developing rat epididymal duct

Hypermethylation-dependent silencing of the gene is achieved by recruiting methyl-CpG binding proteins (MeCPs). Among the MeCPs, MeCP2 is the most abundantly and ubiquitously expressed in various types of cells. We first screened the distribution and expression pattern of MeCP2 in adult and developing rat tissues and found strong MeCP2 expression, albeit rather ubiquitously among normal tissues, in ganglion cells and intestinal epithelium in the small intestine, in Purkinje cells and neurons in the brain, in spermatogonia and in epithelial cells in the epididymal duct of the testis. We then assessed the expression and the methylation pattern of the promoter region of cyclin D1 by immunohistochemistry and sodium bisulfite mapping, and found that cyclin D1 expression in the epididymal duct decreased rapidly during rat development: strong in newborn rats and very weak or almost negative in 7-day-old rats. Mirroring the decrease of cyclin D1 expression, methylated cytosine at both CpG and non-CpG loci in the cyclin D1 promoter was frequently observed in the epididymal duct of 7-day-old rats but not in that of newborn rats. Interestingly, MeCP2 expression also increased concomitant with the increase of methylation. Cyclin D1 expression in the epididymal duct may be efficiently regulated by the epigenetic mechanism of the cooperative increase of MeCP2 expression and promoter methylation.     

Regional distribution of importin subtype mRNA expression in the nervous system: study of early postnatal and adult mouse

Importin-alpha and beta1 mediate the translocation of macromolecules bearing nuclear localization signals across the nuclear pore complex. Five importin-alpha isoforms have been identified in mice and six in human. Some of these importins play an important role in neural activity such as long term potentiation, but the functional differences of each isoform in the CNS are still unclear. We performed in situ hybridization (ISH) using non-isotopic probes to clarify the expression patterns of importin-alpha subtypes (alpha5, alpha7, alpha1, alpha4, alpha3) and importin-beta1 in the mouse CNS of adult and early postnatal stages. The mRNAs of the importin-alpha subtypes and importin beta1 were expressed throughout the CNS with specific patterns; importin-alpha5, alpha7, alpha3, and beta1 showed moderate to high expression levels throughout the brain and spinal cord; importin-alpha4 showed a lack of expression in limited regions; and importin-alpha1 showed a low expression level throughout the brain and spinal cord but with a moderate expression level in the olfactory bulb and reticular system. We also demonstrated that importin-alphas and beta1 mRNAs were predominantly expressed in neurons in the adult mouse brain by using double-labeling fluorescence ISH and immunohistochemistry. Moreover, importin-alphas and beta1 mRNAs were detected throughout the CNS of postnatal mice and were highly expressed in the external granule layer of the cerebellar cortex on postnatal days 0, 4, and 10. This is the first report of importin-alphas and beta1 expression throughout the CNS of adult mice, as well as in the developing brain, including cell type specific localization.     

40例急性胰腺炎患者血清淀粉样蛋白A及炎症因子的变化

目的 :观察血清SAA、CRP、IL - 6、IL - 8及SIL - 2R在急性胰腺炎病程中的变化 ,探讨SAA在急性胰腺炎发生发展中的辅助诊断及疾病严重度评价的实用价值。方法 :SAA、CRP定量检测 :采用乳胶增强速率散射比浊法。IL - 6、IL - 8、SIL - 2R水平采用ABC -ELISA法检测。结果 :急性重症胰腺炎患者五项指标显著高于急性轻症胰腺炎及正常对照组 (p <0 0 1 )。同时血清SAA水平与CRP、IL - 6、IL - 8水平呈显著相关 (p <0 0 1 )。急性轻症胰腺炎组SAA、CRP、IL - 6水平高于正常对照组 (p <0 0 5 )。 结论 :联合检测血清SAA、CRP、IL - 6、IL - 8水平对判断急性重症胰腺炎的严重程度及提示急性胰腺炎坏死性形成具有重要参考价值。在急性胰腺炎的病程中SAA的检测价值优于CRP。     

Regional adaptations in PSD-95, NGFI-A and secretogranin gene transcripts related to vulnerability to behavioral sensitization to amphetamine in the Roman rat strains

Genetically selected for high or low two-way active avoidance, Roman high-avoidance (RHA) and Roman low-avoidance (RLA) rats differ in their central dopaminergic activity, sensation/novelty- and substance-seeking profiles. These animals are, therefore, well suited to identify anatomical and neurochemical concomitants of behavioral sensitization, a phenomenon linked to addictive liability. We submitted inbred RHA (RHA-I), inbred RLA (RLA-I) and Sprague-Dawley-OFA (SD-OFA) rats to a sensitization regimen with amphetamine and studied the behavioral response to an amphetamine challenge after a 2-week withdrawal period. The expression patterns of nerve growth factor inducible clone A (NGFI-A), secretogranin, post-synaptic density protein of 95 Kd (PSD-95), prodynorphin and proenkephalin mRNA were also analyzed using in situ hybridization, after the challenge with amphetamine. RHA-I rats showed stronger sensitization than SD-OFA rats. RLA-I rats did not show sensitization but were hyper-reactive to amphetamine. Expression of behavioral sensitization in RHA-I rats activated secretogranin and PSD-95 mRNA in the nucleus accumbens core. On the other hand, high induction of NGFI-A mRNA in the central amygdala was observed in RLA-I rats when they experienced amphetamine for the first time in the challenge. Our results reveal that 1) the acute locomotor response to amphetamine does not predict vulnerability to behavioral sensitization and 2) differences in vulnerability to sensitization may involve distinctive cellular adaptations at particular brain locations which may be related to addictive vulnerability.     

Human serum mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of levels in body fluids and identification of two forms in serum

We developed an ELISA for human serum MASP-1, a Cls-like serine protease which is known to function in C4 and C2 activation. We then determined MASP-1 levels in 1063 sera from normal Japanese subjects ranging in age from 3 to 100 years, as well as in certain body fluids using this assay. Individual serum MASP-1 levels ranged from 148 to 12–83 μg/ml, with a normal frequency distribution pattern. The arithmetic mean ± s.d. of MASP-1 levels in serum was 6–27 ± 185 μg/ml, whereas levels of MASP-1 in cerebrospinal fluid and in urine were almost undetectable. When the mean ± s.d. of serum MASP-1 was calculated for each age group (10 year range) and values were then compared, the age group consisting of 3–9-year-olds (7–54 ± l-39;μ/ml) was found to have the highest value. When MASP-1 was measured in cord blood, it was shown that levels were already as high as those of 3–9-year-olds. The serum MASP-1 level was found to be as strongly dependent on age as is the serum MBL level. MASP-1 and MBL are thought to play an active part in immunity in younger people. It was found that the serum level of MASP-1 was much higher than that of MBL, and the major portion of human serum MASP-1 appeared to exist in the circulation as a form unbound to MBL.     

Quantitative radial immunodiffusion assay for serum amyloid A protein

A radial immunodiffusion assay for serum amyloid A protein (SAA) using a commercially available antiserum is described. Serum is applied untreated to 1% agarose gels prepared in 0.02 M barbitone buffer, pH 8.6, containing 40 g/l polyethylene glycol 6000. Incubation is carried out overnight at 37 degrees C. The assay combines the advantages of simplicity, rapidity, specificity and stability, and avoids the hazards associated with the previously described radioimmunoassays. The method has sufficient sensitivity to measure SAA in the majority (99%) of normal subjects, and confirms the behaviour of SAA as a very sensitive acute phase reactant in inflammatory disease. The method is ideally suited to the rapid processing of a large number of samples.     

Changes in Laminin Chain Expression in Pre- and Postnatal Rat Pituitary Gland

Cell–matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5–15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.     

Developmental expression and distribution of opioid receptors in zebrafish

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.     

Immunohistochemical distribution of serum proteins in living mouse heart with In vivo cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods.     

Upregulation of tPA/plasminogen proteolytic system in the periphery of amyloid deposits in the Tg2576 mouse model of Alzheimer's disease

Although the tissue plasminogen activator (tPA)/plasminogen/plasmin proteolytic system is thought to modulate the catabolism of amyloid-β (Aβ), in vivo evidence remains insufficient. In the brain of human amyloid precursor protein transgenic Tg2576 mice, we found co-accumulation of tPA and plasminogen at the periphery of compact amyloid deposits, mainly Aβ42-cored plaques, as well as in the walls of blood vessels with cerebral amyloid angiopathy (CAA). This tPA/plasminogen system contained high levels of proteolytic activity. High levels of tPA were also found in reactive astrocytes with increased Aβ42 expression, whereas plasminogen was found only in neurons. When the brain sections of Tg2576 mice were treated with both tPA and plasminogen, levels of thioflavin-S fluorescence, congophilicity and birefringence in the compact amyloid plaques were significantly reduced, and the ultrastructure of Aβ42-fibrils was disrupted. These results suggest that the assembled Aβ42 may promote upregulation of the tPA/plasminogen proteolytic system, which can modulate the deposition of amyloid plaques in vivo.     

Behavioral and molecular changes in the mouse in response to prenatal exposure to the anti-epileptic drug valproic acid

Experiments in rodents have indicated that maternal valproic acid (VPA) exposure has permanent adverse effects upon neurological and behavioral development. In humans, prenatal exposure to VPA can induce fetal valproate syndrome, which has been associated with autism. The present study examined mouse pups exposed in utero to VPA, measuring physical development, olfactory discrimination, and social behavior as well as expression of plasticity-related genes, brain derived neurotrophic factor (BDNF) and NMDA receptor subunits NR2A and NR2B. VPA-exposed mice showed delayed physical development, impaired olfactory discrimination, and dysfunctional pre-weaning social behavior. In situ hybridization experiments revealed lower cortical expression of BDNF mRNA in VPA animals. These results support the validity of the VPA mouse model for human autism and suggest that alterations in plasticity-related genes may contribute to the behavioral phenotype.     

Context-driven cocaine-seeking in abstinent rats increases activity-regulated gene expression in the basolateral amygdala and dorsal hippocampus differentially following short and long periods of abstinence

In this study, the expression patterns of zif268 and activity-regulated cytoskeleton-associated gene (arc) were investigated in the basolateral amygdala (BLA) and dorsal hippocampal (dHPC) subregions during context-induced drug-seeking following 22 h or 15 d abstinence from cocaine self-administration. Arc and zif/268 mRNA in BLA and dHPC increased after re-exposure to the cocaine-paired chamber at both timepoints; however, only the BLA increases (with one exception-see below) were differentially affected by the presence or absence of the cocaine-paired lever in the chamber. Following 22 h of abstinence, arc mRNA was significantly increased in the BLA of cocaine-treated rats re-exposed to the chamber only with levers extended, whereas following 15 d of abstinence, arc mRNA in the BLA was increased in cocaine-treated rats returned to the chamber with or without levers extended. In contrast, zif268 mRNA in the BLA was greater in cocaine-treated rats returned to the chamber with levers extended vs. levers retracted only after 15 d of abstinence. In the dentate gyrus (DG) following 22 h of abstinence, zif268 mRNA was greater in rats returned to the chamber where levers were absent regardless of drug treatment whereas arc mRNA was increased in CA1 (cell bodies and dendrites) and CA3 only in cocaine-treated groups. Following 15 d of abstinence, arc mRNA was significantly greater in CA1 and CA3 of both cocaine-treated groups returned to the chamber than in those placed into a familiar, non-salient alternate environment; however, only in CA1 cell bodies the cocaine context-induced increases significantly greater than in yoked-saline controls. In contrast, zif/268 mRNA in all dHPC regions was significantly greater in both cocaine-treated groups returned to the cocaine context than in the cocaine-treated group returned to an alternative environment or saline-treated groups. These data suggest that the temporal dynamics of arc and zif268 gene expression in the BLA and dHPC encode different key elements of drug context-induced cocaine-seeking.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

Up-regulation of macrophage colony-stimulating factor (M-CSF) and migration inhibitory factor (MIF) expression and monocyte recruitment during lipid-induced glomerular injury in the exogenous hypercholesterolaemic (ExHC) rat

Although macrophages play an important role in lipid-induced glomerular injury, we know little of the mechanisms by which hyperlipidaemia induces monocyte recruitment. This study investigated the role of M-CSF and macrophage MIF in monocyte recruitment during the development of lipid-induced glomerular injury in the susceptible ExHC rat strain. Groups of five ExHC rats were fed a high cholesterol diet (HCD) containing 3% cholesterol, 0.6% sodium cholate and 15% olive oil, and killed after 3 days, 1, 2 or 6 weeks. Control animals were killed on day 0 or after 6 weeks on a normal diet. Animals were hypercholesterolaemic 3 days after the induction of the HCD, but showed no change in plasma triglycerides over the 6-week period. Glomerular macrophage accumulation was first evident at 1–2 weeks and increased up to week 6, when macrophage-derived foam cells were seen in almost all glomeruli, and segmental lesions and mild proteinuria were also evident. Combined in situhybridization and immunohistochemistry staining demonstrated that, coincident with the induction of hypercholesterolaemia on day 3, there was marked up-regulation of M-CSF and MIF mRNA expression by intrinsic glomerular cells (mostly mesangial cells and podocytes) which preceded monocyte recruitment. There was a highly significant correlation between the number of M-CSF and MIF-positive cells and glomerular macrophage accumulation over the 6-week period. Although some glomerular macrophages and foam cells exhibited M-CSF and MIF expression, the major source of these molecules was intrinsic glomerular cells. No local macrophage proliferation was observed during the development of glomerular lesions. In conclusion, hypercholesterolaemia caused marked up-regulation of M-CSF and MIF expression by intrinsic glomerular cells, which correlated with monocyte recruitment and the development of lipid-induced glomerular injury. This is the first study to implicate local synthesis of MIF in the pathogenesis of lipid-induced lesions.