Developmental patterns of mST3GaIV mRNA expression in the mouse:In situ hybridization using DIG-labeled RNA probes

mST3GalV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GalV (CMP-NeuAc: lactosylceramide alpha2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GalV in mice, we investigated the spatial and temporal expression of mST3GalV mRNA during the mouse embryogenesis [embryonic (E) days; E9, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from E9 and E11 were positive for mST3GalV mRNA. On E13, mST3GalV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3Gal V which was a quite similar to that of E13. In this stage, mST3GalV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GalV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.     

In situ hybridization histochemistry as a method to assess GABA(A) receptor subunit mRNA expression following chronic alprazolam administration.

Previous work in our laboratory has demonstrated region-specific effects for chronic alprazolam on binding and function at the GABA(A) receptor. The present study evaluated regional changes in mRNA expression of several subunits of the GABA(A) receptor following chronic alprazolam administration that might underlie these effects. Mice received alprazolam (2 mg/kg/day) or vehicle via subcutaneously implanted osmotic pumps for 1, 7, 14 or 28 days. In situ hybridization histochemistry was performed on tissue sections using [35S]dATP oligonucleotide probes corresponding to the alpha1 and gamma2 subunits of the GABA(A) receptor. Specific hybridization was clearly demonstrated and alpha1 subunit mRNA expression in frontoparietal cortex (layers II-IV) on day 1 of infusion was reduced in animals receiving alprazolam compared to vehicle. On subsequent days, there were no alterations in the levels of alpha1 subunit mRNA in the frontoparietal cortex, hippocampus or dentate gyrus. Expression of gamma2 subunit mRNA was increased on day 1 in the frontoparietal cortex (layer VI), hippocampus and dentate gyrus. mRNA expression was also increased in the dentate gyrus on day 28 of infusion. Comparison of the present study with the results of chronic treatment with other benzodiazepines clearly demonstrates that the pattern of mRNA subunit alterations obtained is both treatment- and region-specific. This makes a definitive conclusion regarding benzodiazepines and their interactions with GABA(A) receptors difficult at best.     

In situ hybridization studies of UDP-glucuronosyltransferase UGT1A6 expression in rat testis and brain

UDP-glucuronosyltransferases (UGTs), in addition to their role in overall pharmacokinetics, play important roles in local protection of cells against toxins and in the control of endogenous receptor ligands. UGT1A6, which conjugates planar phenols, appears to be expressed in many organs, but information on cell-specific expression in these organs is controversial or absent. Therefore, a non-isotopic in situ hybridization method was developed and applied to localize UGT1A6 expression in rat testis and brain. It was found that UGT1A6 is expressed in Sertoli cells and spermatogonia of rat testis and in brain neurons, in particular in hippocampal pyramidal cells and Purkinje cells of the cerebellum.     

In situ detection of prolactin receptor mRNA and apoptotic cell death in mouse uterine tissues with adenomyosis

Prolactin receptor (PRLR) mRNA was visualized by in situ hybridization in adenomyotic uteri of mice with ectopic pituitary grafting. The signals of PRLR mRNA were mainly detected in the smooth muscle cells of myometrium and the epithelial cells of uterine glands and lumen. In the uteri with adenomyosis, the expression intensity was stronger than that in the normal uteri of control mice. This might reflect the higher serum prolactin (PRL) levels in the mouse with pituitary graft, as reported previously. Electron microscopic observation showed that apoptotic cell death occurred in certain cells in the myometrium bearing adenomyotic changes. Such changes were rarely observed in the normal uteri. The relevance of these findings to the development of adenomyosis was examined.     

The effects of isolation rearing on glutamate receptor NMDAR1A mRNA expression determined by in situ hybridization in Fawn hooded and Wistar rats

Rats reared in social isolation exhibit a syndrome of behavioral and biochemical effects indicative of enhanced mesolimbic dopamine (DA) function. The precise nature of the neurodevelopmental changes that produce this state are unknown but result in enhanced DA neurotransmission in the nucleus accumbens (NAC). It was hypothesized that this may be the indirect result of chronic changes in glutamate NMDA receptor function. The same prediction has been made for Fawn hooded (FH) rats that exhibit some of the characteristic effects of isolation-reared rats when compared to Wistar rats. Therefore, mRNA levels of the NMDAR1A receptor subunit were determined by in situ hybridization and were quantified in the striatum, hippocampus and prefrontal cortex of FH and Wistar rats. Isolation rearing alone was not found to have an effect on the expression of NMDAR1A, while FH rats had reduced levels across most brain regions examined. In some areas of the striatum and prefrontal cortex, this effect was greater in FH isolates than in FH socials, while in the hippocampus, the opposite was observed.     

Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization.

We used in situ hybridization to examine organ- and cell type-specific constitutive and 3-methylcholanthrene (3MC)-inducible cytochrome P450 (CYP)1A1 and CYP1A2 mRNA expression in various tissues of the C57BL/6N mouse. In situ hybridization was carried out 10 hr after the mice had received intraperitoneal 3MC, or vehicle alone. We detected levels of 3MC-induced CYP1A1 mRNA in: liver (centrilobular, more so than periportal, regions); lung (Clara Type II cells much more than Type I epithelial cells); brain, especially endothelial cells lining the vascular surface of the choroid plexus; the digestive tract (duodenum > jejunum > ileum > colon > esophagus > stomach--in particular, the villous epithelium, plus cells surrounding glands in the lamina propria); renal corpuscles of the kidney; the ovary (medulla more so than cortex); and the endothelial cells of blood vessels throughout the animal. Constitutive CYP1A1 mRNA was not detectable by in situ hybridization in any of these tissues. In contrast, constitutive CYP1A2 mRNA was measurable in liver, and 3MC-inducible CYP1A2 mRNA was observed only in liver, lung, and duodenum (having cell-type locations similar to those of CYP1A1); the other above-mentioned tissues were negative for CYP1A2 mRNA. These data demonstrate the striking differences in tissue- and cell type-specific expression between the two members of the mouse Cypla subfamily. Because of the ubiquitous nature of 3MC-inducible CYP1A1 throughout the animal rather than just "portals of entry," these results support our hypothesis that CYP1A1, induced by particular endogenous signals in various tissues and cell types, might participate in one or more critical life processes--in addition to its well-established role of metabolism of polycyclic hydrocarbons, certain drugs, and other environmental pollutants.     

Metformin raises hydrogen sulfide tissue concentrations in various mouse organs

BackgroundThe epidemic of diabetes mellitus type 2 forces to intensive work on the disease medication. Metformin, the most widely prescribed insulin sensitizer, exerts pleiotropic actions on different tissues by not fully recognized mechanisms. Hydrogen sulfide (H₂S) is involved in physiology and pathophysiology of various systems in mammals and is perceived as a potential agent in the treatment of different disorders. The interaction between biguanides and H₂S is unknown. The aim of the study is to assess the influence of metformin on the H₂S tissue concentrations in different mouse organs.MethodsAdult SJL female mice were administered intraperitoneally 100 mg/kg b.w. per day of metformin (group D1, n = 6) or 200 mg/kg b.w. per day of metformin (group D2, n = 7). The control group (n = 6) received physiological saline. The measurements of the free and acid-labile H₂S tissue concentrations were performed with Siegel spectrophotometric modified method.ResultsThere was a significant progressive increase in the H₂S concentration along with the rising metformin doses as compared to the control group in the brain (D1 by 103.6%, D2 by 113.5%), in the heart (D1 by 11.7%, D2 by 27.5%) and in the kidney (D1 by 7.1%, D2 by 9.6%). In the liver, massive H₂S accumulation was observed in the group D1 (increase by 420.4%), while in the D2 group only slight H₂S level enhancement was noted (by 12.5%).ConclusionOur experiment has shown that metformin administration is followed by H₂S tissue concentrations increase in mouse brain, heart, kidney and liver.     

内皮素受体mRNA在心衰患者心脏中的表达

目的 研究内皮素受体ＥＴＡ、ＥＴＢ在心力衰竭时的变化及与心衰病因的联系。方法 用Ｎｏｒｔｈｅｒｎ Ｂｌｏｔ方法检测正常人、冠心病和扩张专肌病所致心力衰竭病人心脏 左心室组织ＥＴＡ、ＥＴＢｍＲＮＡ。结果 冠心病组ＥＴＡｍＲＮＡ明显高于对照组（Ｐ＝０．００７）及扩张型心肌病组（Ｐ＝０．０１５）。扩张型心肌病组与正常对照组相比无显著差别;冠心病组和扩张型心肌病组ＥＴＡｍＲＮＡ水平与相应心脏指数、左室射血分数及舒张末期左心室内径均无显著相关性;各组间ＥＴＢｍＲＮＡ无明显差别。结论 心力衰竭时ＥＴＡ的合成表达与引起心衰的病因密切相关。冠心病性心衰病人ＥＴＡ合成表达明显增加,缺氧可能是刺激ＥＴＡ合成表达的最重要因素;ＥＴＢ介导的血管作用在心力衰竭中不表现重要的病理生理作用。     

IL—2,IL—2R,IL—6mRNA在自身免疫性甲状腺病甲状腺组织中的原位表达

本实验分别用Ｄｉｇ标记的ＩＬ－２、ＩＬ－２Ｒ、ＩＬ－６ｃＲＮＡ探针在３７例自身免疫性甲状腺疾病患者甲状腺石蜡切片上行原位杂交，结果显示患者甲状腺浸润细胞中有ＩＬ－２、ＩＬ－２Ｒ、ＩＬ－６ｍＲＮＡ的异常表达，其分布与病理损伤部位一致，这种异常表达是引起甲状腺内免疫功能紊乱的重要因素之一。     

细胞原位杂交法检测肿瘤细胞中C-myc基因的表达

本文采用生物素标记的C-myc基因作探针,通过细胞原位分子杂交技术,对人舌癌、膀胱癌和正常细胞中C-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比,舌癌和膀胱癌细胞中C-myc基因异常高度表达。     

A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5-ht7 receptor in rat brain.

1. Receptor autoradiography and in situ hybridization histochemistry have been used to delineate the distribution of the 5-ht7 receptor and its mRNA in rat brain. Receptor autoradiographic studies were performed using [3H]-5-carboxamidotryptamine (5-CT) as the radioligand. The binding characteristics of the masking compounds were determined in Cos-7 cells transfected with a panel of 5-HT receptor subtype cDNAs, including the rat 5-ht7 cDNA. In situ hybridization studies were carried out with 35S-labelled oligonucleotide probes to the rat 5-ht7 mRNA. 2. Specific binding of [3H]-5-CT was observed in many areas of the rat brain. Following co-incubation with 1 microM ergotamine, this binding was completely eliminated. After addition of the masking ligands, [3H]-5-CT binding remained in layers 1-3 of cortex, septum, globus pallidus, thalamus, hypothalamus, centromedial amygdala, substantia nigra, periaquaductal gray, and superior colliculus. Addition of the antagonist, methiothepin, to the incubation regimen eliminated most of the remaining [3H]-5-CT binding in the brain, with the exception of the globus pallidus and substantia nigra. 3. The 5-ht7 mRNA was discretely localized in rat brain. The most intense hybridization signals were observed over the thalamus, the anterior hippocampal rudiment, and over the CA3 region of the hippocampus. Other regions containing hybridization signals included the septum, the hypothalamus, the centromedial amygdala and the periaquaductal gray. The regions exhibiting a modest receptor binding signal after methiothepin incubation, the globus pallidus and the substantia nigra, contained no 5-ht7 hybridization signals, suggesting a non-5-ht7 subtype in these two related structures. 4. The distribution of the 5-ht7 receptor and its mRNA is suggestive of multiple roles for this novel 5-HT receptor, within several brain systems. The limbic system (centromedial amygdala, anterior hippocampal rudiment, hypothalamus) is particularly well-represented, indicating a potential role for the 5-ht7 receptor in affective processes.     

Localization of somatostatin (SRIF) SSTR-1, SSTR-2 and SSTR-3 receptor mRNA in rat brain by in situ hybridization

In situ hybridization histochemistry was performed to analyse the distribution of the messenger RNA (mRNA) of three putative somatostatin (SRIF) receptors in rat brain, using oligonucleotide probes derived from the cDNA coding for SSTR-1, SSTR-2, and SSTR-3 receptors.SSTR-1 signals were found in layers VVI of the cerebral cortex, in primary olfactory cortex, taenia tecta, subiculum, entorhinal cortex, granular layer of the dentate gyrus, amygdala and cerebellar nuclei. Signals for SSTR-2 were found in the frontal cerebral cortex (layers IV, V and VI), taenia tecta, claustrum, endopiriform nucleus, locus coeruleus, medial habenula, subiculum, granular cell layer of the dentate gyrus and amygdala. High levels of SSTR-3 hybridization were found in the olfactory bulb, primary olfactory cortex, islands of Calleja, medial habenula, amygdala, granular layer of the dentate gyrus, various thalamic and pontine nuclei and in the granular and Purkinje cell layers of the cerebellum.The distribution of the hybridization signals of the oligoprobes is consistent with the labelling of specific SRIF binding sites in rat brain. Especially, SSTR-2 and SSTR-1 oligos seem to label regions in which SS-1 and SS-2 receptors, respectively, have been previously characterized in autoradiographical studies. The situation is less clear with SSTR-3 mRNA, since SRIF binding in adult rats is usually low or absent in cerebellum, although some cerebellar nuclei appear to be labelled in the adult. The localization of SSTR-1, SSTR-2 and SSTR-3 mRNAs suggests that SRIF receptor subtypes in rat brain show profound differences in their distribution and are involved in a variety of central, in addition to neuroendocrine, functions.Monique Rigo, who contributed very significantly to this work, died tragically on January 21, 1993 Correspondence to: D. Hoyer at the above address     

Constitutive mRNA expression of various glutathione S-transferase isoforms in different tissues of mice.

Glutathione S-transferase (Gst) enzymes are instrumental in protecting cellular macromolecules against electrophiles and products of oxidative stress. Of interest primarily to pharmacologists and toxicologists is the ability of these enzymes to metabolize cancer chemotherapeutic drugs, insecticides, herbicides, and carcinogens. Thus, constitutive expression of Gsts might determine a tissue's ability to handle certain forms of chemical stress. In the present study, the constitutive mRNA expression of 19 different Gst enzymes was investigated in 14 different tissues in mice. The information obtained from the present study could be distilled into a few generalized principles: in all tissues examined, multiple isoforms of Gst were constitutively expressed; several isoforms, such as Gstk1, Gstm1, Gstm4, Gstm6, and Gstt1, were expressed in most of the tissues studied; at least five Gst isoforms were highly expressed in the gonads, about three in heart, and at least one in brain (Gstm5). Gender differences in the expression of various Gst isoforms were pronounced. With a few exceptions, most of the Gst isoforms expressed in kidney showed higher expression in females than males; the same trend was observed for heart and gonads. At least eight Gst isoforms showed very high expression in stomach. This was a unique finding in the current study because drug-metabolizing enzymes that are highly expressed in the gastrointestinal (GI) tract tend to have the highest expression in small intestine with low or no expression in the stomach. In summary, most Gst isoforms are most highly expressed in the GI tract and liver, which strongly suggests an important role of many Gst isoforms in detoxification of ingested xenobiotics.     

Colocalization of neurotransmitters analyzed by in situ hybridization.

In situ hybridization and Northern blot analysis has been used to analyse in some detail the localization and regulation of the messenger molecules adrenaline, noradrenaline and neuropeptide tyrosine (NPY) within cells of the sympathetic nervous system and the adrenal medulla. In the rat adrenal gland, a novel NPY containing population of ganglion cells was found. Synthetic oligonucleotide probes complementary to mRNA coding for the catecholamine synthesizing enzymes phenylethanolamine N-methyltransferase (PNMT), tyrosine hydroxylase (TH) and NPY were used to analyse the regulation of these genes following administration of the catecholamine depleting drug reserpine. Twenty-four hours after a single dose of reserpine, a differential regulation of PNMT, TH and NPY was found. Thus, a dramatic decrease in PNMT mRNA was observed in the adrenal medulla. In contrast, mRNA for both TH and NPY exhibited an increase. Different regulatory mechanisms may thus operate for these three compounds coexisting in chromaffin cells of the adrenal medulla. The regulation of enzymes and peptides was also studied in human sympathetic ganglia. After brief electrical preganglionic stimulation of thoracic ganglia in humans, in situ hybridization was performed with synthetic oligonucleotide probes complementary to TH, dopamine beta-hydroxylase (DBH) and NPY mRNA respectively. A several fold increase in all three mRNAs was found in the principal ganglion cells. The results point to a very rapid regulation of genes involved in signal transmission in the sympathetic nervous system of humans. The results also suggest a novel way to define neuronal projections by visualizing increases in mRNA levels following electrical stimulation.     

Regulation of mRNA expression of xenobiotic transporters by the pregnane x receptor in mouse liver, kidney, and intestine.

Multiple transporter systems are involved in the disposition of xenobiotics and endogenous compounds. The pregnane X receptor (PXR) is a major chemical sensor known to activate the expression of CYP3A/Cyp3a in humans and rodents. The purpose of this study is to systematically determine whether the major xenobiotic transporters in liver, kidney, duodenum, jejunum, and ileum are induced by pregnenolone-16alpha-carbonitrile (PCN), and whether this increase is mediated by the nuclear receptor PXR. In liver, PCN induced the expression of Oatp1a4 and Mrp3 mRNA in wild-type (WT) mouse liver, but not in PXR-null mice. In kidney, PCN did not alter the expression of any drug transporter. In duodenum, PCN increased Abca1 and Mdr1a mRNA expression in WT mice, but not in PXR-null mice. In jejunum and ileum, PCN increased Mdr1a and Mrp2 mRNA, but decreased Cnt2 mRNA in WT mice, but none of these transporters was altered when PCN was administered to PXR-null mice. Therefore, PCN regulates the expression of some transporters, namely, Oatp1a4 and Mrp3 in liver, as well as Abca1, Cnt2, Mdr1a, and Mrp2 in small intestine via a PXR-mediated mechanism.     

In situ hybridization and immunocytochemistry of alpha1-adrenoceptors in human peripheral blood lymphocytes

1 alpha1-Adrenoceptor subtypes were investigated in cytospin centrifuged preparations of human peripheral blood lymphocytes by in situ hybridization and immunocytochemistry. 2 In situ hybridization cytochemistry revealed alpha1A-, alpha1B-, and alpha1D-receptor mRNA in human peripheral blood lymphocytes. Lymphocytes hybridized for alpha1A receptor subtype represented approximately 30% of total lymphocytes, those hybridized for alpha1Beta- and alpha1D-receptor subtypes averaged 42 and 25% of total lymphocytes, respectively. 3 Cytospin centrifuged lymphocytes exposed to anti-alpha1A-, alpha1Beta- or alpha1D-receptor protein antibodies, developed specific immunostaining. Approximately 27% of total lymphocytes were immunoreactive for alpha1A-receptor protein, 40% displayed alpha1B-receptor protein immunoreactivity and 22% alpha1D-receptor protein immunoreactivity. Analysis of percentages as well as of lymphocyte morphology of in situ hybridized and immunolabelled lymphocytes suggests the co-expression of mRNA receptor signal and protein receptor immunostaining in the same lymphocyte. 4 The demonstration of both alpha1-adrenoceptor mRNA and receptor protein subtypes suggests that alpha1-adrenoceptors may have a role in regulating lymphocyte function. 5 The possibility of demonstrating receptor protein immunoreactivity in a small amount of blood, such as that required for preparing cytospin-centrifuged lymphocytes, may stimulate research to evaluate the role of these receptors in lymphocytes and to establish if assessment of lymphocyte alpha1-adrenoceptors may represent a marker of their status in health and disease.     

荧光原位分子杂交技术检测乳腺癌组织中HER-2基因的表达

目的荧光原位杂交(FISH)方法检测乳腺癌组织中人表皮生长因子2(HER-2)的扩增情况,并与免疫组化(IHC)结果相比较,优化乳腺癌HER-2检测方法。方法采用相关质控措施,利用FISH法及IHC法检测乳腺癌HER-2的表达,分析相关性。结果 FISH检测Her-2基因扩增40例,阳性率30.5%(40/131),在IHC(-)标本中,Her-2基因未扩增19例,阴性符合率为95.0%;在IHC(+)标本中,Her-2基因扩增7例,阳性符合率为20.0%;在IHC(++)标本中,Her-2基因扩增15例,阳性符合率26.8%;在IHC(+++)标本中,Her-2基因扩增17例,阳性符合率89.5%。结论 FISH检测准确率较高,IHC阴性和(+++)时与FISH结果一致性较好,可作为治疗依据,IHC(+)和(++)时仅能作初步筛查,明确Her-2情况仍需结合FISH检测。     

Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration

An assay for tyrosine hydroxylase (TH) mRNA by in situ hybridization in combination with immunocytochemistry (ICC) for TH on the same section is described. The in situ hybridization protocol was optimized for [35S]cRNA (complementary RNA, i.e. anti-sense strand) probe concentration and time of hybridization. The specificity of hybridization was measured by several critera. The advantage of measuring grain density versus grains per cell is discussed for quantitation of in situ autoradiography. Finally, the reserpine-induced increase in adrenal TH mRNA was used to validate quantitative aspects of the in situ hybridization technique by comparison with blot hybridization. In contrast to the adrenal, reserpine did not increase TH mRNA in substantia nigra (s. nigra) neurons as measured by either technique.