Effects of two melatonin analogues, S-20098 and S-20928, on melatonin receptors in the pars tuberalis of the rat

Abstract: By using quantitative autoradiography, we studied the effects of two drugs related to melatonin on the 2- ¹²⁵I-melatonin binding in the pars tuberalis (PT) of rats. The drugs tested were two naphthalenic analogues of melatonin, S-20098 ( N -[2-(7-methoxy-l-naphthyl) ethyl] acetamide), an agonist, and S-20928 ( N -[2-(l-naphthyl) ethyl] cyclobutyl carboxamide), a putative antagonist. Melatonin (s.c. and i.p.), S-20098 (s.c), and S-20928 (i.p.) were injected 4 hr before sacrifice. Acute administration of both melatonin and S-20098 decreased melatonin receptor density. In contrast, the putative antagonist S-20928, at a low dose (1 mg/kg), was ineffective on melatonin receptors. It neither affected the 2- ¹²⁵I-melatonin specific binding observed in the control group nor did it prevent the decrease in binding induced by melatonin when injected 5 min before the hormone. At a high dose (10 mg/kg), S-20928 totally blocked the effect of melatonin on melatonin receptor density and induced a decrease in binding capacity as melatonin did when injected alone. These results indicate that in the rat pars tuberalis, the melatonin agonist, S-20098, is able to down-regulate melatonin receptors, whereas S-20928 seems to behave as a partial agonist.     

Daily and photoperiodic 2- 125I-melatonin binding changes in the pars tuberalis of the Syrian hamster (Mesocricetus auratus): Effect of constant light exposure and pinealectomy

Abstract: Using quantitative autoradiography, 2¹²⁵I-melatonin binding was investigated throughout the light/dark cycle in the pars tuberalis (PT) of the pituitary of adult Syrian hamsters kept for 8 weeks either in long or short photoperiod (LP or SP, respectively). Melatonin receptor density in the PT displayed photoperiod dependent daily variations (maximal values in LP). Indeed, in LP, melatonin receptor density underwent strong daily variations with maximal values during the first half of the light period and minimal values at the end of the night. These variations depended on changes in the maximal binding (B_max) without differences in the dissociation constant (K_d). In contrast, PT melatonin receptor density was constant and at a very low level throughout the 1ight:dark cycle in SP exposed animals. Daily PT melatonin receptor density variations of LP exposed animals were abolished by pinealectomy or continuous light exposure. These results show clearly that both at the daily and at the seasonal level the regulation of PT melatonin receptors is strongly dependent on circulating melatonin concentrations in the Syrian hamster, but that other regulatory factors, yet unclarified, might also play a role.     

Effect of melatonin and diazepam on the dissociated circadian rhythm in rats

The main structures involved in the circadian system in mammals are the suprachiasmatic nuclei (SCN) of the hypothalamus. The SCN contain multiple autonomous single-cell circadian oscillators that are coupled among themselves, generating a single rhythm. However, under determined circumstances, the oscillators may uncouple and generate several rhythmic patterns. Rats exposed to an artificially established 22-h light-dark cycle (T22) express two stable circadian rhythms in their motor activity that reflect the separate activities of two groups of oscillators in the morphologically well-defined ventrolateral and dorsomedial SCN subdivisions. In the experiments described in this paper, we studied the effect of melatonin and diazepam (DZP) administration in drinking water on the dissociated components of rat motor activity exposed to T22, to deduce the possible mechanism of these drugs on the circadian system. In order to suppress the endogenous circadian rhythm of melatonin, in some of the rats the pineal gland or the superior cervical ganglia were removed. The results show that melatonin or DZP treatment increased the manifestation of the light-dependent component to the detriment of the manifestation of the non-light-dependent component and that melatonin, but not DZP, shortens the period of the non-light-dependent component. These findings suggest that both DZP and melatonin favor entrainment to external light, and that melatonin could also act on the SCN, producing changes in the period of the circadian cycle.     

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light–dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN′s subregional organization to both photic input processing and rhythmic output control.     

Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse

This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2-[125I]-Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3+/-1.7 fmol/mg protein, n = 5, at ZT2) or at the beginning of the subjective day (48.6+/-2.1 fmol/mg protein, n = 6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35S-labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CTI10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions.     

Long-term exposure of hypothalamic explants to melatonin alters the release of gonadotrophin releasing hormone and the density of melatonin binding sites in the pars tuberalis of the male mink (Mustela vison)

To investigate the action of melatonin on the reproductive system, the effect of prolonged versus short-term exposure to melatonin on the release of gonadotrophin releasing hormone (GnRH) was examined in hypothalamic explants of male mink sacrificed in July, September or November. Mediobasal hypothalamic (MBH) explants including the pars tuberalis (PT) were incubated for 1 night with or without melatonin (10(-8) M) for 8 hr or 16 hr and the release of GnRH was then measured. The next day, the explants were incubated further but in a melatonin free buffer, and the release of GnRH was measured with increasing time. Half of the July and September explants had melatonin binding sites quantified by autoradiography. In November, a 16-hr exposure to melatonin induced a significant increase in the release of GnRH during the night, compared with control or 8-hr melatonin exposure. This increase persisted for at least 45 min after the withdrawal of melatonin, suggesting a stimulatory effect of melatonin on the synthesis of GnRH; this effect was apparent in July, September and November. In September, the density of melatonin binding in the PT was significantly lower in the explants incubated for 16 hr with melatonin, compared with those incubated for 8 hr. Thus, in vitro, a long exposure to melatonin, mimicking a single long night, stimulates the release and synthesis of GnRH in parallel with a decrease in the density of melatonin binding in the PT. These effects seem to depend heavily on the duration of exposure to melatonin.     

Application of the multi-colour FISH to interphase nuclei and metaphase spreads for simultaneous examination of monosomy 7 and trisomies 8 and 11 in acute myelocytic leukaemia (AML)

We have used the multi-colour (three) fluorescence in situ hybridization (FISH) technique based on the ratio labelling for the detection of monosomy 7 and trisomies 8 and 11 in 13 cases of acute myeloid leukaemia (AML). Two out of the 13 AML cases showed monosomy 7 and two out of the remaining cases exhibited trisomy 8 in interphase nuclei. Three of these results were confirmed by metaphase-FISH study. Trisomy 11 was not found either by the interphase FISH study or in the metaphase FISH study. These results demonstrate the potential power of multi-colour FISH using ratio labelling to produce more fluorescein colour in interphase nuclei for the detection of aneuploidies in leukaemia.     

大鼠局灶性脑缺血预处理后生长停滞与DNA损害可诱导基因34表达的变化

目的观察脑缺血预处理(brain ischemia preconditioning,BIP)对大鼠脑缺血再灌注后,生长停滞与DNA损害可诱导基因34(GADD34)mRNA及蛋白表达的影响。方法健康雄性SD大鼠120只,随机分为假手术组、脑缺血组、BIP组,每组40只,后2组行大脑中动脉栓塞法制模后,各组于再缺血后12 h、1、2和3 d 4个时间点观察,每个时间点10只。采用2次线栓法制备大鼠局灶性脑缺血预处理模型,用原位杂交法检测GADD34 mRNA表达,western blot法观察各组GADD34蛋白表达。结果与假手术组比较,脑缺血组12 h GADD34 mRNA及蛋白表达均达高峰,随再灌注时间延长,其表达逐渐下降,但1、2和3 d时仍高于假手术组(P<0.05,P<0.01);BIP组GADD34 mRNA及蛋白表达各时间点均较脑缺血组明显升高(P<0.05,P<0.01)。结论 BIP可诱导GADD34mRNA及蛋白的表达。     

不明原因持续肝功能异常患者肝组织和外周血单个核细胞中HBV DNA表达

采用原位分子杂交技术分别检测随机选择的40例不明原因持续肝功能异常患者外周血单个核细胞和其肝组织中HBV DNA,肝组织中HBV DNA阳性24例,阳性率为58%;外周血单个核细胞中HBV DNA阳性18例,阳性率为45%;两者比较,差异无显著性(x2=2.10,P＞0.05).对肝组织和外周血单个核细胞进行HBVDNA检测,有助于诊断隐性HBV感染.     

Garlic Potyviruses Are Translocated to the True Seeds through the Vegetative and Reproductive Systems of the Mother Plant

Garlic lost its ability to produce true seeds millennia ago, and today non-fertile commercial cultivars are propagated only vegetatively. Garlic viruses are commonly carried over from one generation of vegetative propagules to the other, while nematodes and arthropods further transmit the pathogens from infected to healthy plants. A recent breakthrough in the production of true (botanical) garlic seeds resulted in rapid scientific progress, but the question of whether viruses are transmitted via seeds remains open and is important for the further development of commercial seed production. We combined morpho-physiological analysis, fluorescence in situ hybridization (FISH), and PCR analysis to follow potyvirus localization and translocation within garlic fertile plants and seeds. Spatial distribution was recorded in both vegetative and reproductive organs. We conclude that garlic potyviruses are translocated to the seeds from the infected mother plant during flower development and post-fertilization, while pollen remains virus-free and does not contribute to seed infection. Therefore, the main practical goal for virus-clean seed production in garlic is the careful maintenance of virus-free mother plants. Although garlic pollen is free of potyviral infection, the male parents’ plants also need to be protected from contamination, since viral infection weakens plants, reducing flowering ability and pollen production.     

胃黏膜细胞线粒体DNA不稳定及核内整合与幽门螺杆菌感染有关

目的：线粒体DNA（mtDNA）因缺乏组蛋白保护，且损伤修复系统不健全，容易为幽门螺杆菌（Hp）相关胃炎中氧自由基的重要靶点，为此探讨胃黏膜细胞线粒体DNA不稳及核内整合与Hp感染的关系。方法：采用PCR和Giemsa染色检测Hp；采用限制性片段多态性（PCR－SSCP）和原位杂交方法检测胃黏膜细胞线粒体DNA微卫星不稳定（mtMSI）及核内mtDNA序列。结果：30例胃癌检出mtMSI11例（36．7％），15例肠化中有2例（13．3％），10例异型增生中有2例，10例萎缩性胃炎中有1例检出mtMSI。胃癌细胞核内mtDNA序列的检出率为20．0％（6／30），异型增生为1／10例，肠上皮化生为6．7％（1／15），萎缩性胃炎为1／10例，胃黏膜细胞mtMSI及核内mtDNA序列的检出率在Hp感染组（12／39，8／39）显著高于非Hp感染组（4／36，1／36，P＜0．05）。虽cagA^ 组mtMSI及核内mtDNA序列检出率（10／25，6／25）高于cagA^-组（2／14，2／14），但两组mtMSI及核内mtDNA序列检出率比较，差异并无显著性（P＞0．05）。结论：胃黏膜细胞mtMSI及mtDNA序列核内整合可能与Hp感染有关，并参与胃癌的发生。     

Distribution of hepatitis C virus RNA in the liver and its relation to histopathological changes

Abstract: To investigate a cellular mode of HCV-infection in the liver and its pathological implications in relation to histopathological changes or clinical data, we studied the distribution of HCV-RNA in the livers of 21 patients with HCV-related chronic liver disease (chronic active hepatitis, 14 cases; cirrhosis, 7 cases) using the in situ hybridization technique. In situ hybridization was performed on 4% paraformaldehyde-fixed frozen sections with digoxigenin-labeled DNA probe deduced from the core region of HC–J4. In situ hybridization showed positive signals in the liver specimens of 20/21 cases. The signals were localized in the cytoplasm of hepatocytes. The distribution pattern of positive cells was individually different, whereas the pattern was identical in the right and left lobes. There were no correlations of the HCV-positive cell number with serum aminotransferase levels at biopsy or with genotypes of HCV. The positive hepatocytes were occasionally associated with infiltrating mononuclear cells, and they were sparsely distributed in the area of piecemeal necrosis. These findings suggest that factors such as host immunoreaction to the virus may be more important than its direct cytopathy in the pathogenesis of chronic hepatitis C virus infection.     

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants

BACKGROUND AND AIMS: Resistance is a major challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). We investigated the mechanisms of resistance in patients with progressive GISTs with primary KIT mutations and the efficacy of the kinase inhibitor PKC412 for the inhibition of imatinib-resistant mutants. METHODS: We performed a cytogenetic analysis and screened for mutations of the KIT and PDGFRA kinase domains in 26 resistant GISTs. KIT autophosphorylation status was assessed by Western immunoblotting. Imatinib-resistant GIST cells and Ba/F3 cells expressing these mutant proteins were tested for sensitivity to imatinib and PKC412. RESULTS: Six distinct secondary mutations in KIT were detected in 12 progressive tumors, with V654A and T670I found to be recurrent. One progressive tumor showed acquired PDGFRA -D842V mutation. Amplification of KIT or KIT / PDGFRA was found in 2 patients. Eight of 10 progressive tumors available for analysis showed phosphorylated KIT. Two remaining progressive tumors lost KIT protein expression. GIST cells carrying KIT -del557-558/T670I or KIT -InsAY502-503/V654A mutations were resistant to imatinib, while PKC412 significantly inhibited autophosporylation of these mutants. Resistance to imatinib and sensitivity to PKC412 of KIT -T670I and PDGFRA -D842V mutants was confirmed using Ba/F3 cells. CONCLUSIONS: This study shows the high frequency of KIT/PDGFRA kinase domain mutations in patients with secondary resistance and defines genomic amplification of KIT / PDGFRA as an alternative cause of resistance to the drug. In a subset of patients, cancer cells lost their dependence on the targeted tyrosine kinase. Our findings show the sensitivity of the imatinib-resistant KIT -T670I and KIT -V654A and of PDGFRA -D842V mutants to PKC412.     

Interphase fluorescent in situ hybridization deletion analysis of the 9p21 region and prognosis in childhood acute lymphoblastic leukaemia (ALL): results from a prospective analysis of 519 Nordic patients treated according to the NOPHO-ALL 2000 protocol

Interphase fluorescent in situ hybridization (FISH) was applied on diagnostic BM smears from 519 children with acute lymphoblastic leukaemia (ALL) in order to establish the frequency and prognostic importance of 9p21 deletion in children enrolled in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) - 2000 treatment protocol. Among the patients, 452 were diagnosed with B-cell precursor (BCP)-ALL and 66 with T-ALL. A higher incidence of 9p21 deletions was found in T-ALL (38%) compared to BCP-ALL (15·7%). Homozygous deletions were found in 19·7% of T-ALL and 4·0% of BCP-ALL; hemizygous deletions were found in 18·2% and 11·7% respectively. In our series, 9p21 deletions were detected in all age groups with a steady rise in the frequency with age. There was no significant difference in outcome between cases with or without 9p21 deletion or between cases with hemi- or homozygous deletions of 9p21. In conclusion, in this large series of childhood ALL deletion of 9p21 was not associated with worse prognosis. However, interphase FISH deletion analysis of 9p21 could be used as a first step to detect unfavourable subtle cytogenetic aberrations such as the dic(9;20) rearrangement.     

慢性乙型肝炎患者肝组织中巨噬细胞移动抑制因子的表达及其对炎症活动的影响

目的:检测巨噬细胞移动抑制因子(macrophage migrationinhibitory factor,MIF)在正常肝和慢性乙型肝炎(chronic hepatitis B,CHB)肝组织中表达的差异,探讨MIF的表达与CHB炎症活动及病毒载量的关系。方法:取CHB40例为实验组(HBeAg阳性CHB28例,HBeAg阴性CHB12例),正常肝组织20例为对照组,用免疫组化和原位杂交技术检测两组中MIF和MIFmRNA的表达,并测定患者ALT和AST的水平及HBV DNA载量。结果:MIF和MIF mRNA在实验组中的阳性表达例数明显高于对照组,两组相比差异有显著性意义(P<0.05);实验组肝组织中MIF和MIF mRNA表达程度与ALT和AST呈正相关,1相似文献   

Resistance to Fas (APO-1/CD95)-mediated apoptosis and expression of Fas ligand in esophageal cancer: the Fas counterattack

The mechanisms by which esophageal tumors escape immunologic recognition and clearance are only partly understood at the molecular level. Esophageal cancers have been shown to evade host recognition by down-regulation of antigen presentation and production of immunosuppressive factors. Recently, two independent reports have shown that esophageal tumor cells abundantly express Fas ligand (FasL) in vivo. As the triggering agonist for Fas receptor (Fas or APO-1/CD95)-mediated apoptosis of lymphocytes, FasL normally plays immune down-regulatory roles, including activation-induced cell death of T and B cells, as well as maintaining immune privilege in certain organs. Fas ligand expressed by esophageal cell lines has been shown to induce apoptosis of cocultured Fas-sensitive lymphoid cells in vitro. FasL expression by esophageal carcinomas in vivo has been associated with significantly reduced tumor-infiltrating lymphocytes (TILs) in FasL-positive tumor nests, concomitant with significantly increased TIL apoptosis in these nests. These studies support a 'Fas counterattack' mechanism of immune escape in esophageal cancer. By expressing functional Fas ligand, esophageal cancer cells can deplete antitumor lymphocytes by inducing apoptosis. To express functional FasL, esophageal carcinomas also acquire molecular mechanisms to resist autocrine Fas-mediated apoptosis of tumor cells.     

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.