Diurnal pattern of clock gene expression in the hypothalamus of the newborn rabbit

In the European rabbit (Oryctolagus cuniculus) nursing acts as a strong non-photic synchronizer of circadian rhythmicity in the newborn young. Rabbits only nurse for a few minutes once every 24 h and previous studies have shown that the pups, blind at birth, display endogenous circadian rhythms in behavior and physiology entrained by this regular daily event. As a further step toward understanding the neural organization of the rabbit's early circadian system, we investigated the expression of clock genes in the suprachiasmatic nucleus of the hypothalamus (SCN; the principal circadian pacemaker in adult mammals) across the pups' 24-h day. We used 43 pups from seven litters maintained in constant darkness and entrained non-photically by nursing at the same time each day until P7. After nursing on day 7, pups were killed in the dark at 3-h intervals so as to obtain eight groups (n=5-6 pups/group) distributed evenly across the 24 h before the next scheduled nursing. Profiles in the expression of the clock genes Per1, Per2, Cry1 and Bmal1 were determined using in situ hybridization in brain sections through the hypothalamus at the level of the SCN. We report for the first time: 1) that Per1, Per2, Cry1 and Bmal1 are all expressed in the SCN of the newborn rabbit, 2) that the expression of Per1, Per2 and Bmal1 but not Cry1 shows diurnal rhythmicity similar to that in adult mammals, and 3) that the expression of Per1, Per2 and Bmal1 is consistent with the strong entraining effect of nursing found in previous studies. Unexpectedly, and contrasting somewhat to the pattern in the SCN, we also found diurnal rhythmicity in the expression of Cry1 and Bmal1 but not of Per1 in the anterior ventromedial hypothalamic nucleus. Overall, our findings suggest that the SCN is a functional part of the newborn rabbit's circadian system and that it can be entrained by non-photic cues associated with the mother's daily nursing visit.     

Circadian profile and photic regulation of clock genes in the suprachiasmatic nucleus of a diurnal mammal Arvicanthis ansorgei

The molecular mechanisms of the mammalian circadian clock located in the suprachiasmatic nucleus have been essentially studied in nocturnal species. Currently, it is not clear if the clockwork and the synchronizing mechanisms are similar between diurnal and nocturnal species. Here we investigated in a day-active rodent Arvicanthis ansorgei, some of the molecular mechanisms that participate in the generation of circadian rhythmicity and processing of photic signals. In situ hybridization was used to characterize circadian profiles of expression of Per1, Per2, Cry2 and Bmal1 in the suprachiasmatic nucleus of A. ansorgei housed in constant dim red light. All the clock genes studied showed a circadian expression. Per1 and Per2 mRNA increased during the subjective day and decreased during the subjective night. Also, Bmal1 exhibited a circadian expression, but in anti-phase to that of Per1. The expression of Cry2 displayed a circadian pattern, increasing during the late subjective day and decreasing during the late subjective night. We also obtained the phase responses to light for wheel-running rhythm and clock gene expression. At a behavioral level, light was able to induce phase shifts only during the subjective night, like in other diurnal and nocturnal species. At a molecular level, light pulse exposure during the night led to an up-regulation of Per1 and Per2 concomitant with a down-regulation of Cry2 in the suprachiasmatic nucleus of A. ansorgei. In contrast, Bmal1 expression was not affected by light pulses at the circadian times investigated. This study demonstrates that light exposure during the subjective night has opposite effects on the expression of the clock genes Per1 and Per2 compared with that of Cry2. These differential effects can participate in photic resetting of the circadian clock. Our data also indicate that the molecular mechanisms underlying circadian rhythmicity and photic synchronization share clear similarities between diurnal and nocturnal mammals.     

Circadian gene expression predicts patient response to neoadjuvant chemoradiation therapy for rectal cancer

Preoperative neoadjuvant chemoradiation therapy may be useful in patients with operable rectal cancer, but treatment responses are variable. We examined whether expression levels of circadian clock genes could be used as biomarkers to predict treatment response. We retrospectively analyzed clinical data from 250 patients with rectal cancer, treated with neoadjuvant chemoradiation therapy in a single institute between 2011 and 2013. Gene expression analysis (RT-PCR) was performed in tissue samples from 20 patients showing pathological complete regression (pCR) and 20 showing non-pCR. The genes analyzed included six core clock genes (Clock, Per1, Per2, Cry1, Cry2 and Bmal1) and three downstream target genes (Wee1, Chk2 and c-Myc). Patient responses were analyzed through contrast-enhanced pelvic MRI and endorectal ultrasound, and verified by histological assessment. pCR was defined histologically as an absence of tumor cells. Among the 250 included patients, 70.8% showed regression of tumor size, and 18% showed pCR. Clock, Cry2 and Per2 expressions were significantly higher in the pCR group than in the non-pCR group (P<0.05), whereas Per1, Cry1 and Bmal1 expressions did not differ significantly between groups. Among the downstream genes involved in cell cycle regulation, c-Myc showed significantly higher expression in the pCR group (P<0.05), whereas Wee1 and Chk2 expression did not differ significantly between groups. Circadian genes are potential biomarkers for predicting whether a patient with rectal cancer would benefit from neoadjuvant chemoradiation therapy.     

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland.

Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with beta-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation.     

Expression of Circadian <Emphasis Type="Italic">Per1</Emphasis> and <Emphasis Type="Italic">Per2</Emphasis> Genes in the Liver and Breast Tumor Tissues of HER2/neu Transgenic Mice of Different Age

The expression of Per1, Per2, and Cry1 circadian genes in the liver and breast tumors were studied by real-time PCR in FVB/N mice of different age transfected with HER-2/neu gene. The expression of Per1 and Per2 genes in breast tumor tissue decreased in comparison with their expression in the lever. The expression of these genes decreased with age in both the liver and tumor tissue.     

Effect of lipopolysaccharide on circadian clock genes <Emphasis Type="Italic">Per2</Emphasis> and <Emphasis Type="Italic">Bmal1</Emphasis> in mouse ovary

In mammals, circadian rhythms are associated with multiple physiological events. The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on circadian systems in the ovary. Immature female mice were received an intra-peritoneal injection of equine chorionic gonadotropin (eCG) and LPS. Total RNA was collected from the ovary at 6-h intervals throughout a 48 h of experimental period. The expression of the circadian genes period 2 (Per2) and brain and muscle ARNT-like 1 (Bmal1) such as circadian genes was measured by quantitative PCR. Although expression of Per2 and Bmal1 in the ovary did not display clear diurnal oscillation, LPS suppressed the amplitude of Per2 expression. Additionally, LPS inhibited the expression of cytochrome P450 aromatase (CYP19) and luteinizing hormone receptor (LHr) genes in the ovary of eCG-treated mice. Our data suggest that Per2 may be associated with the inhibition of CYP19 and LHr expression by LPS in the ovaries of immature mice.     

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.     

模拟微重力对NIH3T3细胞近日节律基因的影响

目的:研究模拟微重力对NIH3T3细胞近日节律基因表达水平的影响。方法:NIH3T3细胞按照模拟微重力的天数分为5组,RT-PCR检测节律基因mRNA的相对表达水平。结果:实验结果显示五组样品Per1、Per2、Cry1、Bmal1、Clock的相对表达水平存在显著性差异。Per1和Per2基因mRNA的相对表达水平在模拟微重力的第2天、第3天较0天显著升高(P0.05),Per2、Cry1和Clock基因mRNA的相对表达水平在模拟微重力的第4天较其他四组显著降低(P0.05)。结论:近日节律基因的相对表达水平在模拟微重力第2、3天升高,第4天后降低。模拟微重力影响近日节律基因的表达且具有时间依赖性。     

Effects of ethanol on microsomal drug metabolism in aging female rats. III. In vivo

The effects of aging on ethanol inhibition of zoxazolamine metabolism in vitro and in vivo were studied in female Fischer 344 rats aged 4, 14 and 26 months. Zoxazolamine hydroxylase activity in freshly-isolated liver microsomes decreased significantly with age (1.88 +/- 0.32, 1.49 +/- 0.30 and 0.74 +/- 0.18 nmol/min per mg protein in young-adult, middle-aged and old rats, respectively). A substantial inhibition of zoxazolamine hydroxylation occurred in the presence of 40 mM ethanol. The extent of inhibition was the same in microsomes from all three age groups. The effect of aging on the duration of zoxazolamine paralysis in vivo reflected the effect of aging on zoxazolamine metabolism in vitro. Mean duration of paralysis following a standard 50 mg/kg dose of zoxazolamine increased significantly as a function of aging (0.5, 2.9 and 4.7 h in young-adult, middle-aged and old rats, respectively). Administration of ethanol (1.2 g/kg) 10 min before zoxazolamine treatment prolonged the duration of zoxazolamine paralysis in young-adult and middle-aged rats by about 2 to 2.5 h, but ethanol pretreatment did not affect paralysis time in old rats. Thus, the inhibitory effect of ethanol on zoxazolamine metabolism in vivo appeared to be attenuated in old age.     

Dark pulse resetting of the suprachiasmatic clock in Syrian hamsters: behavioral phase-shifts and clock gene expression

In mammals, the circadian clock in the suprachiasmatic nuclei (SCN) is mainly synchronized to photic cues provided by the daily light/dark cycle. Phase-shifts produced by light exposure during the night are correlated with rapid induction of two clock genes, Per1 and Per2, in the SCN. Nonphotic stimuli such as behavioral and pharmacological cues, when presented during the subjective day, induce behavioral phase-advances and a down-regulation of Per1 and Per2 expression in the SCN. When applied during the subjective day, dark pulses in continuous light also produce phase-advances. These phase-shifting effects have been interpreted as reflecting either a photic image mirror, nonphotic cues, or a combination of both. Here we evaluated in Syrian hamsters housed in constant light how dark pulses applied in late subjective day affect levels of Per1, Per2 and Cry1 mRNA. Four-hour dark pulses with no access to a wheel produced 1.2+/-0.4 h phase-advances of locomotor activity rhythm while control manipulation induced non-significant shifts (0.1+/-0.2 h). Dark pulses transiently down-regulated Per1 and Per2 mRNA levels in the SCN by 40 and 20% respectively, while the levels of Cry1 mRNA remained unaffected. In behaviorally split hamsters in which Per oscillations were asymmetric between the left and right sides of the SCN, dark pulses reduced Per expression in the half-SCN with high Per. This study shows that exposure during the late subjective day to dark pulses independent of wheel-running have nonphotic-like effects on the SCN clock at both behavioral and molecular levels.