Electroacupuncture Intervention Regulates Circadian Rhythms by Down-regulating Per Gene Expression in Hypothalamic Suprachiasmatic Nucleus of Hepatocellular Carcinoma Mice / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the rhythm of running-wheel activity of hepatocellular carcinoma (HCC) mice and the expression of Per 1 and Per 2 (circadian rhythm genes) in the hypothalamic suprachiasmatic nucleus (SCN), so as to investigate its mechanism underlying regulating circadian rhythm. METHODS: A total of 108 male C 57 BL / 6 J mice were randomly divided into control, HCC model and EA groups which were further assigned to six zeitbeger (environmental light-dark cycle) time (ZT) point (ZT 0, ZT 4, ZT 8, ZT 12, ZT 16 and ZT 20) subgroups. The HCC model was established by injection of H 22 cancer cell (abdominal 3rd generation, 10 µL) suspension into the larger live lobe. Mice of the control group received saline injection of the liver lobe. EA (2 Hz/15 Hz, 0.2 mA) was applied to bilateral "Ganshu" (BL 18) and "Zhiyang" (GV 9) for 15 min, once daily for 10 days. Mice of the control and model groups received the same binding-fixing to those of the EA group. Circadian running-wheel activity of 12 h∶12 h light darkness (LD) cycle (activity onset and acrophase of actogram, amplitude or peak of periodogram) was recorded by using ClockLab (ACT-500) software and analyzed by MATLAB (R 2007 b) before and after EA treatment. The pathological changes of liver cells were observed under light microscope after sectioning and H．E. staining. The expression levels of Per 1 mRNA and Per 2 mRNA in the liver tissues were determined by fluorogenic quantitative real time-PCR. RESULTS: (1) Following modeling, the amplitude of periodogram of running-wheel activity was significantly lowered at ZT 0, ZT 4, ZT 8, ZT 12, ZT 16, and ZT 20 relevant to the control group (P0.05). (2) The expression levels of Per 1 mRNA and Per 2 mRNA in the SCN were significantly up-regulated at the 6 time-points in the model group relevant to the control group (P<0.05), and obviously down-regulated at ZT 8 after EA intervention relevant to the model group (P<0.05)．. CONCLUSION: EA can benignly regulate the rhythm of running-wheel activity of HCC mice, which may be closely related to its effect in down-regulating the expression of circadian rhythm genes Per 1 and Per 2 in the SCN.

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.

Melatonina inhibe la respuesta de cortisol a ACTH: posible mediación a través del gen reloj Per1 / Melatonin inhibition of the cortisol response to ACTH may be exerted through period circadian protein homolog 1 (Per1)

Background: Circadian cortisol production results from the interaction of the circadian production of ACTH, the autonomic nervous system and intrinsic factors within the gland. An additional regulator is the neuro-hormone melatonin. In human adrenal gland cultures, melatonin inhibited ACTH stimulated cortisol production and Per1 mRNA expression. ACTH actions on the adrenal involve early and late responses. Aim: To investigate the effects of melatonin on the time course of ACTH stimulated cortisol production and of Per1 expression in the lamb adrenal gland. Material and Methods: Adrenal glands and plasma of five newborn lambs were obtained. Adrenal glands were cut in 15 mg explants. Three of these explants were stored for RNA extraction. The rest of explants were using in different culture protocols with ACTH and melatonin. Results: Lambs had an in vivo a circadian variation in plasma cortisol and in adrenal Per1 expression. In vitro, ACTH stimulated an early and late increase in cortisol production and an early increase in Per1 expression reaching a maximum at 3 hours of treatment. Melatonin inhibited the early Per1 response to ACTH without affecting the early ACTH stimulated cortisol production. However, melatonin inhibited the late response of cortisol production to ACTH. Conclusions: The inhibitory actions of melatonin on Per1 response to ACTH may contribute to the inhibitory effects of melatonin on adrenal steroidogenic response to ACTH.

Suprachiasmatic nucleus slices induce molecular oscillations in fibroblasts / 中华行为医学与脑科学杂志

Objective To study whether suprachiasmatic nucleus (SCN) slices are able to induce the molecular oscillations in NIH/3T3 fibroblast. Methods SCN slices from 10-day-old SD rat and NIH/3T3 cells were co-cultured in a serum-free condition. 24h mRNA profiles of Per1 and Rev-Erbα were measured in NIH/3T3 cells using real-time PCR. Results After co-cultured for 6 days, ten SCN slices can induce the significant daily oscillation of Per1 and Rev-Erba mRNA expression in NIH/3T3 cells (P<0.01). The peak time Rev-erbα and Per1 were at CT5 and CT11 respectively. Rev-Erbα oscillations were significant even with two SCN slices and 2 days co-culture (P<0.05). In contrast, Per1 expression fluctuation was not observed until more than 6 days of co-culture and with six SCN slices (P=0.031). Conclusion Diffusible signals release from SCN slices can regulate molecular rhythms in cultured fibroblasts. Rev-Erbα and Per1 don't start to oscillate at the same time, and Rev-Erbα is more sensitive to SCN signal.

Determination of Genomospecies and Characterization of Antimicrobial Resistance of Multi-drug Resistant Acinetobacter spp. Isolates

Acinetobacter species are non-fermentative Gram-negative coccobacilli and they have emerged as important nosocomial pathogens which are associated with the significant multidrug resistance in recent years. Carbapenem-resistant A. baumannii (CRAB) and pandrug-resistant A. baumannii (PDRAB) were reported in 1991 and 1998, respectively. Fiftyeight isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005 were investigated for the existence of CRAB, PDRAB, extended-spectrum beta-lactamase (ESBL)-producing Acinetobacter and examined for their phenotypic and genotypic characteristics. Genomospecies of Acinetobacter species were determined by amplified rDNA restriction analysis (ARDRA) and antimicrobial susceptibility test was performed with 13 kinds of antimicrobial agents. Metallo-beta-lactamase (MBL) producers were screened by modified hodge test and confirmed by imipenem-EDTA disk synergy test. Detection of blaIMP-1, blaVIM-2, blaTEM, and blaPER-1 was performed by PCR. Genomic DNAs were analyzed by pulsed-field gel electrophoresis (PFGE). Among 58 isolates of Acinteobacter species, 40 isolates were identified as genospecies 2 (A. baumannii), 9 were 13TU, 5 were A. phenon 6/ct, and 4 were Acinetobacter genospecies 3 by ARDRA. Thirteen isolates were confirmed as MBL-producers and blaIMP-1 and blaVIM-2 were carried by 5 and 8 isolates of them, respectively. MBL-producers were mostly 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 and they were susceptible to ciprofloxacin and ampicillin-sulbactam. BlaPER-1 was carried by thirteen isolates and 12 isolates of them were PDRAB showing resistance to all antimicrobial agents tested, including ceftazidime, cefepime, aztreonam, ciprofloxacin, amikacin, gentamicin, ampicillin-sulbactam, and imipenem. In conclusion, most MBL-producers belonged to 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 which were susceptible to ciprofloxacin and ampicillin-sulbactam, whereas 12 of 13 PER-1-producers were PDRAB originated from the same clone.

Determination of Genomospecies and Characterization of Antimicrobial Resistance of Multi-drug Resistant Acinetobacter spp. Isolates

Acinetobacter species are non-fermentative Gram-negative coccobacilli and they have emerged as important nosocomial pathogens which are associated with the significant multidrug resistance in recent years. Carbapenem-resistant A. baumannii (CRAB) and pandrug-resistant A. baumannii (PDRAB) were reported in 1991 and 1998, respectively. Fiftyeight isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005 were investigated for the existence of CRAB, PDRAB, extended-spectrum beta-lactamase (ESBL)-producing Acinetobacter and examined for their phenotypic and genotypic characteristics. Genomospecies of Acinetobacter species were determined by amplified rDNA restriction analysis (ARDRA) and antimicrobial susceptibility test was performed with 13 kinds of antimicrobial agents. Metallo-beta-lactamase (MBL) producers were screened by modified hodge test and confirmed by imipenem-EDTA disk synergy test. Detection of blaIMP-1, blaVIM-2, blaTEM, and blaPER-1 was performed by PCR. Genomic DNAs were analyzed by pulsed-field gel electrophoresis (PFGE). Among 58 isolates of Acinteobacter species, 40 isolates were identified as genospecies 2 (A. baumannii), 9 were 13TU, 5 were A. phenon 6/ct, and 4 were Acinetobacter genospecies 3 by ARDRA. Thirteen isolates were confirmed as MBL-producers and blaIMP-1 and blaVIM-2 were carried by 5 and 8 isolates of them, respectively. MBL-producers were mostly 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 and they were susceptible to ciprofloxacin and ampicillin-sulbactam. BlaPER-1 was carried by thirteen isolates and 12 isolates of them were PDRAB showing resistance to all antimicrobial agents tested, including ceftazidime, cefepime, aztreonam, ciprofloxacin, amikacin, gentamicin, ampicillin-sulbactam, and imipenem. In conclusion, most MBL-producers belonged to 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 which were susceptible to ciprofloxacin and ampicillin-sulbactam, whereas 12 of 13 PER-1-producers were PDRAB originated from the same clone.

Emergence of CTX-M-12, PER-1 and OXA-30 beta-Lactamase-Producing Klebsiella pneumoniae / 대한임상미생물학회지

BACKGROUND: The aim of this study was to determine a nation-wide prevalence of Ambler class A and D extended-spectrum-lactamases (ESBL) in Klebsiella pneumoniae isolates in Korea. METHODS: During the period of April to May 2005, 189 isolates of K.pneumoniae were collected from 11 Korean hospitals. Antimicrobial susceptibilities to ceftazidime and cefotaxime were tested by the disk diffusion method, and ESBL production was determined by double-disk synergy test. Determinants of ceftazidime or cefotaxime-resistance were transferred to Escherichia coli J53 (azide-resistant) by transconjugation. Genotypes of class A and D ESBL genes were determined by PCR amplification and sequencing. RESULTS: One hundred-sixty isolates of K.pneumoniae showed positive results in double-disk synergy test. The most prevalent ESBL was SHV-12 (n=148). Also detected were genes encoding ESBLs including TEM-52 (n=1), SHV-2a (n=2), CTX-M-3 (n=15), CTX-M-9 (n=6), CTX-M-12 (n=2), CTX-M-14 (n=9), CTX-M-15 (n=1), PER-1 (n=1), GES-5 (n=3), and OXA-30 (n=2) beta-lactamases. CONCLUSION: With the emergence of CTX-M-12, PER-1, and OXA-30 beta-lactamases, the ESBLs in K.pneumoniae isolates are becoming more diverse in Korea.

PER-1 Type Extended-spectrum ?-Lactamases Produced by Gram-negative Bacilli / 中华医院感染学杂志

OBJECTIVE To investigate the prevalence of PER-1 type ESBLs-producing Enterobacteriaceae and their antimicrobial-resistance profile. METHODS Totally 167 strains of Escherichia coli, 154 strains of Klebsiella pneumoniae and 67 strains of Enterobacter cloacae were tested. All of them were isolated from all kinds of clinical specimens in four hospitals of Nanchang city from Aug 2003 to Jun 2004. Antimicrobial susceptibility test(AST) was determined by K-B disk diffusion test. double-disk test(CTX, CTX/CA and CAZ, CAZ/CA)and three- dimension extract test were used to determine ESBLs. PER-1 genes were amplified by PCR. The products of PCR were sequenced to identify its ?-lactamase encoding gene. RESULTS Sixteen isolates were PER-1 gene positive. The PER-1 gene positive rates of E. coli, K. pneumoniae and E. cloacae were 3.6%, 3.2% and 9.0% , respectively . The antimicrobial-resistant rates of isolates harboring blaPER-1 to cefotaxime, ceftaxidime, amikacin , cefoxitin , cefepime and imipenem were 100%,100%, 56%, 94%, 31% and 0%, respectively. CONCLUSIONS blaPER-1 is detected in E. coli, K. pneumoniae and E. cloacace, and has diffused in Enterobacteriaceae . Imipenem and amikacin may be used to treat the infection caused by bacteria harboring blaPER -1.

Prevalence of PER-1 Extended-Spectrum beta-Lactamase-Producing Clinical Isolates of Acinetobacter baumannii in a University Hospital, Busan, Korea / 대한임상미생물학회지

BACKGROUND: In recent years, Acinetobacter baumannii isolates acquired resistance to cefepime have increased significantly. The aim of this study was to survey the prevalence of PER-1 extendedspectrum beta -lactamase (ESBL)-producing A. baumannii isolates in a University Hospital, Busan, Korea. METHODS: Antimicrobial susceptibilities were tested by the disk diffusion method, and double disk synergy test was performed for screening of ESBL-production. MICs were determined by agar dilution method. The isoelectric points of beta -lactamases were determined by isoelectric focusing. Transferability of cefepime-resistance were tested by conjugation. blaPER-1 and blaPER-2 alleles were detected by PCR, and the DNA sequences of amplified products were determined by using the dideoxy-chain termination method. RESULTS: Among 51 clinical isolates of A. baumannii intermediate or resistant to cefepime, 10 isolates (19.6%) showed positive results in double disk synergy test. PCR-based experiments detected blaPER-1 gene in all the 10 isolates. All the isolates contained three beta -lactamase bands: pI 5.3, 7.9, and 9.4. MICs of ampicillin, piperacillin, cephalothin, cefoxitin, cefoperazone, ceftazidime, cefotaxime, cefepime, and aztreonam were >256 mg/L, respectively, and them of imipenem were 8-16 mg/L. CONCLUSION: The prevalence of PER-1-producing A. baumannii strains in Busan was less than that of in Seoul. But an outbreak of infection caused by this strain in an intensive care unit shows that spread of PER-1-producing A. baumannii strains can be anticipated in a near future. Prevention of hospital infection by these resistant microorganisms are needed.