Circadian Rhythms in Heart Rate,Motility, and Body Temperature of Wild‐type C57 and eNOS Knock‐out Mice Under Light‐dark,Free‐run,and After Time Zone Transition

The nitric oxide (NO) system is involved in the regulation of the cardiovascular system in controlling central and peripheral vascular tone and cardiac functions. It was the aim of this study to investigate in wild‐type C57BL/6 and endothelial nitric oxide synthase (eNOS) knock‐out mice (eNOS‐/‐) the contribution of NO on the circadian rhythms in heart rate (HR), motility (motor activity [MA]), and body temperature (BT) under various environmental conditions. Experiments were performed in 12∶12 h of a light:dark cycle (LD), under free‐run in total darkness (DD), and after a phase delay shift of the LD cycle by ?6 h (i.e., under simulation of a westward time zone transition). All parameters were monitored by radiotelemetry in freely moving mice. In LD, no significant differences in the rhythms of HR and MA were observed between the two strains of mice. BT, however, was significantly lower during the light phase in eNOS‐/‐ mice, resulting in a significantly greater amplitude. The period of the free‐running rhythm in DD was slightly shorter for all variables, though not significant. In general, rhythmicity was greater in eNOS‐/‐ than in C57 mice both in LD and DD. After a delay shift of the LD cycle, HR and BT were resynchronized to the new LD schedule within 5–6 days, and resynchronization of MA occurred within 2–3 days. The results in telemetrically instrumented mice show that complete knock‐out of the endothelial NO system—though expressed in the suprachiasmatic nuclei and in peripheral tissues—did not affect the circadian organization of heart rate and motility. The circadian regulation of the body temperature was slightly affected in eNOS‐/‐ mice.     

Endothelial nitric oxide is not involved in circadian rhythm generation of blood pressure: experiments in wild-type C57 and eNOS knock-out mice under light-dark and free-run conditions

Endothelial nitric oxide synthase knock out mice (eNOS-/-) are mildly hypertensive in comparison to wild-type (WT) mice. Hypertension in eNOS-/- mice is partly the result of an increase in peripheral resistance due to the absence of the vasodilatory action of NO. No data are available for these animals regarding the 24 h blood pressure profile under the 12:12 h light-dark cycle (LD) and constant dark (DD) conditions. Therefore, this study aimed to investigate by radiotelemetry the circadian rhythms in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of six eNOS-/- mice and five wild-type mice under LD and DD. Data were collected beginning 3 wks after operation (implantation of sensor) for 2 wks under LD and for another 2 wks thereafter under DD. Our results show that eNOS-/- mice were hypertensive under all experimental conditions. SBP and DBP were significantly higher by about 15% in eNOS-/- mice. No differences were found in the pattern of the circadian rhythms, rhythmicity, or period lengths during LD or DD. The genetic deletion of eNOS seems to lead to higher SBP and DBP, but the circadian blood pressure pattern is still preserved with higher values during the night (active phase) and lower values during the daytime (rest phase). Thus, endothelial-derived NO plays an important role in the regulation of vascular tone and haemodynamics, but it is not important for the circadian organization of SBP and DBP.     

Effect of NO synthase inhibition on cardiovascular circadian rhythms in wild-type and eNOS-knock-out mice

Cardiovascular functions (blood pressure [BP], heart rate [HR]) were radiotelemetrically studied in endothelial nitric oxide synthase (NOS) knock-out mice (eNOS-/-) and their wild type C57BL/6 (WT) controls. Studies were performed with and without inhibition of the NOS with the non-specific inhibitor N(omega)-Nitro-L-Arginin-Methylester (L-NAME). Six eNOS-/-and five WT mice, kept under a light:dark schedule of 12:12 h (lights on 07:00 h), were treated with L-NAME in tap water containing different concentrations (94, 282, and 940 mg/kg) each for three days. Under control conditions, the eNOS-/-mice are mildly hypertensive in comparison to WT. L-NAME increased systolic [SBP] and diastolic [DBP] blood pressures in WT mice to the levels of eNOS-/-mice after two days of L-NAME application with no dose-dependency, whereas L-NAME had no effects on SBP and DBP in eNOS-/-mice. In neither mouse strain were the circadian rhythms in BP and HR affected by drug treatment. The similarity of the 24 h BP profiles in eNOS-/-and L-NAME-treated WT mice support the notion that only the enothelial NOS and not other NOS isoenzymes are of importance for hypertension in the knock-out mouse strain.     

Cyanidin-3-Glucoside Modulates hsa_circ_0001345/miRNA106b/ATG16L1 Axis Expression as a Potential Protective Mechanism against Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common form of malignancy in the liver. Autophagy was found to have a significant effect in controlling HCC. Anthocyanins, which are naturally occurring pigments in a variety of fruits and vegetables, have been thoroughly documented to be involved in a variety of bioactive activities and are widely employed for their antioxidant capabilities. Cyanidin-3-glucoside (C3G) extracted from Morus alba L. has promising antioxidant and anti-tumour activities. The current study aims to examine the protective action of C3G against hepatocellular carcinoma through the investigation of the autophagy protein ATG16L1 expression along with its related RNA molecules (hsa_circ_0001345 and miRNA106b) in Wistar rats. In vivo precancerous lesions (PCL) were induced using diethylnitrosamine (DEN) and acetamidofluorene (2-AAF). Rats were treated with C3G (10, 15, and 20 mg/kg; 4 times weekly) for 112 days (16 weeks). Liver function tests, alfa fetoprotein, ATG16L1 expression, hsa_circ_0001345, and miRNA106b differential expression were examined. Liver sections were examined by histological and immunohistochemical approaches. The current study’s findings indicated that C3G administration protects against the negative effects of DEN-2-AAF on liver functions and liver histopathological sections, which nominated C3G as a potential prophylactic agent against HCC.     

Experimental studies on the afferent innervation of the cricothyroid muscle in dogs

The cricothyroid muscle in dogs received branches from two independent nerves, namely the external ramus of the cranial laryngeal nerve and the pharyngeal branch of the vagus. Classical spindles are infrequent in the muscle. Atypical forms of sensory endings were identified. Two end-plates were frequently met with on a single extrafusal fibre. Sectioning of the external ramus of the cranial laryngeal nerve was followed by degeneration of spindles. Intact axons detected up to 6 months after operation are probably derived from the pharyngeal branch of the vagus. Chromatolytic changes occurred in the ipsilateral dorsal vagal nucleus and the capsulated ganglion at the entry of the nerve into the muscle. Chromatolysis occurred in the intramuscular ganglion cell rows and in neurons of the ipsilateral nodose ganglion. Morphological alterations were more pronounced in the ipsilateral medial column of the nucleus ambiguus. No changes were observed in the somata of the mesencephalic nucleus.     

Blockade of Nitric Oxide Overproduction and Oxidative Stress by <Emphasis Type="Italic">Nigella sativa</Emphasis> Oil Attenuates Morphine-Induced Tolerance and Dependence in Mice

The effects of Nigella sativa oil on morphine-induced tolerance and dependence in mice and possible mechanism(s) of these effects were investigated, for the first time, in this study. Repeated administration of Nigella sativa oil (4 ml/kg, p.o.) along with morphine (5 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of morphine to mice or by administration of naloxone to morphine-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments were inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of the oil on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25 mg/kg). Similarly, concurrent i.p. administration of the NO synthase inhibitors; L-N (G)-nitroarginine methyl ester (10 mg/kg), aminoguanidine (20 mg/kg) and 7-nitroindazole (25 mg/kg) or the antioxidant, N-acetylcysteine (50 mg/kg) enhanced this inhibitory effect of the oil. On the other hand, this effect was antagonized by concurrent i.p. administration of the nitric oxide precursor, L-arginine (300 mg/kg). These results provide evidence that Nigella sativa oil, through inhibition of morphine-induced NO overproduction and oxidative stress, appears to have a therapeutic potential in opioid tolerance and dependence.     

NMR-based metabolomic analysis of wild,greenhouse, and in vitro regenerated shoots of <Emphasis Type="Italic">Cymbopogon schoenanthus</Emphasis> subsp. <Emphasis Type="Italic">proximus</Emphasis> with GC–MS assessment of proximadiol

Cymbopogon schoenanthus subsp. proximus is a wild plant distributed in subtropical and east Africa extending from the north to the southern parts of Egypt. Widely used in folk medicine, it is the source of the diuretic sesquiterpene proximadiol. Nuclear magnetic resonance metabolomic analysis of polar extracts of shoots from wild, greenhouse, somatic embryos, and direct and indirect organogenic in vitro cultures was carried out. Metabolic profiling yielded 39 compounds, of which common metabolites were 15 (38.4%). Unique metabolites were trehalose (2.5%) in the wild plants, 2-hydroxylisobutyrate, galactarate and tyrosine (7.6%) in indirect organogenic shoots. Tartrate was found only in direct regenerated shoots (2.5%). Metabolites identified in greenhouse and embryogenic shoots showed no unique compounds. Multivariate analysis revealed significant differences between all tested shoots. 4-aminobutyrate, alanine, glutamine, glucose, fructose, and sucrose were the most significantly different metabolites. Proximadiol was identified and quantitatively measured from the non-polar extract of different types of shoots using gas chromatography and mass spectrometry (GC–MS). Concentrations ranged from 3.6 ± 0.03 to 198.6 ± 7.2 µg/100 mg dry weight in regenerated shoots from somatic embryogenesis and in wild plant shoots, respectively. Direct organogenesis yielded the highest in vitro concentration (20.3 ± 0.5 µg/100 mg dry weight). This study reported the metabolic profiling of C. schoenanthus polar extract and identified primary metabolites that are unique to the wild type and shoots regenerated from different in vitro cultures. Proximadiol was quantified and the in vitro culture system yielding the highest concentration relative to the wild plant was identified.     

Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K-12

Using a multicopy plasmid in which the tac promoter has been placed in front of the dam gene of Escherichia coli K-12, we show that levels of DNA adenine methylase activity are correlated with the spontaneous mutation frequency.