DAY-NIGHT VARIATION IN THE IN VITRO CONTRACTILITY OF AORTA AND MESENTERIC AND RENAL ARTERIES IN TRANSGENIC HYPERTENSIVE RATS*

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

A SEX DETERMINING MECHANISM IN THE CLOSTERIUM EHRENBERGII (CHLOROPHYTA) SPECIES COMPLEX1

Homozygous mt^?/mt^? diploid clones of the Closterium ehrenbergii Menegh. ex Ralfs species complex were obtained by hypertonic treatment from minus vegetative cells, and mating type segregation ratios in the F₁ progeny of “triploid” zygospores between wild type mt⁺ haploid and mt^?/mt^? homozygous diploui were analyzed. The ratio of plus to minus individuals was 1:4.8, and the ratio of the pairs of opposite mating types to those of minus mating type was 1:2.1. The results clearly show that mt^? is dominant to mt⁺ and that the mating type inheritance in these zygospores follows the triploid-like pattern. The validity of our assumption that the two mating types are determined by one genetic factor (mt^? allele dominant) was confirmed in B₁ progeny analyses as well. The results suggest that this sex determining mechanism is working effectively in the C. ehrenbergii species complex, in which several biological species have evolved through polyploidization.     

MT2-like melatonin receptor modulates amplitude receptor potential in visual cells of crayfish during a 24-hour cycle

Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2 h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT₂ melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT₂ receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [¹²⁵I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [¹²⁵I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (K_d) of 1.1 nM and a total number of binding sites (B_max) of 6 fmol/mg protein at CT 6, and a K_d of 1.46 nM and B_max of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT₂-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells.     

SEASONAL VARIATION IN HEART RATE VARIABILITY IN ASTHMATIC CHILDREN

Asthma is a “seasonal disease” with symptoms either aggravated by environmental changes during specific seasons or prevalent at certain times of the year for other reasons. We examined whether the heart rate variability (HRV) of asthmatic children changes by season. The HRV during a portion of one night (00:00–04:00) and day (12:00–16:00) and the entire 24h period (00:00–24:00) during each of the four seasons was analyzed. The data of 95 children with asthma and 106 healthy children, as controls, were assessed. In children with asthma during the 24h period, seasonal variation in the low-frequency (LF) band (0.04–0.15 Hz) and the high-frequency (HF) band (0.15–0.4 Hz) were detected (HF: F = 6.81, p =.0003; LF: F = 4.18, p =.008. The HF value in the summer was significantly higher than in autumn and spring (Scheffe test: autumn vs. summer, s = 4.46, p<.001; spring vs. summer, s = 2.86, p<.05), while the LF value in autumn was significantly lower than in summer (s = 3.42, p <.01). In the control group, no seasonal variation in HF, LF, or LF/HF was detected. The findings infer the HRV, a surrogate measure of autonomic nervous system function, of asthmatic children is more susceptible to seasonal changes brought about by either endogenous annual rhythms or environmental weather phenomena. (Chronobiology International, 17(4), 503–511, 2000)     

SEXUALITY AND UNIPARENTAL INHERITANCE OF CHLOROPLAST DNA IN THE ISOGAMOUS GREEN ALGA ULVA COMPRESSA (ULVOPHYCEAE)1

Ulva compressa L. is a heterothallic macroalga considered to be in the early evolutionary stage between isogamy and anisogamy. Two genetic lines of this species, each consisting of gametophytes with opposite mating types, were collected on the coasts of Ehime and Iwate prefectures: MGEC‐1 (mt⁺) and MGEC‐2 (mt^?) from Ehime, and MGEC‐5 (mt⁺) and MGEC‐6 (mt^?) from Iwate. Their relative gamete sizes (i.e., cell volumes) do not correspond to their mating types: MGEC‐6 (19.8 μm³) > MGEC‐1 (18.6 μm³) > MGEC‐5 (17.0 μm³) > MGEC‐2 (10.1 μm³). The pattern of organelle inheritance is an important sexual characteristic in many eukaryotes. We therefore investigated the relationship between gamete size and the inheritance of chloroplast DNA (cpDNA). Polymorphisms between the cpDNA of the two lines were used as markers. We found a 24 bp insertion between psbF and psbL, and the substitution of a StyI site (from C CAAGG to T CAAGG) in the intergenic region between petD and accD. Two interline crosses (MGEC‐1 × MGEC‐6 and MGEC‐2 × MGEC‐5) produced 42 and 38 zygotes, respectively. PCR and PCR–RFLP analyses showed that the cpDNA of the mt⁺ gametes was consistently inherited in both crosses. The cpDNA is inherited from one parent only, and it depends not on gamete size but on being mt⁺. The cpDNA was observed during crossing and in the zygotes 6 h after mating. In 6% of the zygotes, the cpDNA derived from the mt^? gametes disappeared 3–4 h after mating. Preferential digestion of the cpDNA in the zygote’s mt^? gamete may form the basis for uniparental inheritance of cpDNA.     

INCREASED SENSITIVITY TO LIGHT-INDUCED MELATONIN SUPPRESSION IN PREMENSTRUAL DYSPHORIC DISORDER

Increased sensitivity to light-induced melatonin suppression characterizes some, but not all, patients with bipolar illness or seasonal affective disorder. The aim of this study was to test the hypothesis that patients with premenstrual dysphoric disorder (PMDD), categorized as a depressive disorder in Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), have altered sensitivity to 200 lux light during mid-follicular (MF) and late-luteal (LL) menstrual cycle phases compared with normal control (NC) women. As an extension of a pilot study in which the authors administered 500 lux to 8 PMDD and 5 NC subjects, in the present study the authors administered 200 lux to 10 PMDD and 13 NC subjects during MF and LL menstrual cycle phases. Subjects were admitted to the General Clinical Research Center (GCRC) in dim light (<50 lux) to dark (during sleep) conditions at 16:00?h where nurses inserted an intravenous catheter at 17:00?h and collected plasma samples for melatonin at 30-min intervals from 18:00 to 10:00?h, including between 00:00 and 01:00?h for baseline values, between 01:30 and 03:00?h during the 200 lux light exposure administered from 01:00 to 03:00?h, and at 03:30 and 04:00?h after the light exposure. Median % melatonin suppression was significantly greater in PMDD (30.8%) versus NC (?0.2%) women (p?=?.040), and was significantly greater in PMDD in the MF (30.8%) than in the LL (?0.15%) phase (p?=?.047). Additionally, in the LL (but not the MF) phase, % suppression after 200 lux light was significantly positively correlated with serum estradiol level (p ?=? .007) in PMDD patients, but not in NC subjects (p?>?.05). (Author correspondence: bparry@ucsd.edu)     

Older Poor-Sleeping Women Display a Smaller Evening Increase in Melatonin Secretion and Lower Values of Melatonin and Core Body Temperature Than Good Sleepers

Melatonin concentration and core body temperature (CBT) follow endogenous circadian biological rhythms. In the evening, melatonin level increases and CBT decreases. These changes are involved in the regulation of the sleep-wake cycle. Therefore, the authors hypothesized that age-related changes in these rhythms affect sleep quality in older people. In a cross-sectional study design, 11 older poor-sleeping women (aged 62–72 yrs) and 9 older good-sleeping women (60–82 yrs) were compared with 10 younger good-sleeping women (23–28 yrs). The older groups were matched by age and body mass index. Sleep quality was assessed by the Pittsburgh Sleep Quality Index questionnaire. As an indicator of CBT, oral temperature was measured at 1-h intervals from 17:00 to 24:00?h. At the same time points, saliva samples were collected for determining melatonin levels by enzyme-linked immunosorbent assay (ELISA). The dim light melatonin onset (DLMO), characterizing the onset of melatonin production, was calculated. Evening changes in melatonin and CBT levels were tested by the Friedman test. Group comparisons were performed with independent samples tests. Predictors of sleep-onset latency (SOL) were assessed by regression analysis. Results show that the mean CBT decreased in the evening from 17:00 to 24:00?h in both young women (from 36.57°C to 36.25°C, p < .001) and older women (from 36.58°C to 35.88°C, p < .001), being lowest in the older poor sleepers (p < .05). During the same time period, mean melatonin levels increased in young women (from 16.2 to 54.1 pg/mL, p < .001) and older women (from 10.0 to 23.5 pg/mL, p < .001), being lowest among the older poor sleepers (from 20:00 to 24:00?h, p < .05 vs. young women). Older poor sleepers also showed a smaller increase in melatonin level from 17:00 to 24:00?h than older good sleepers (mean?±?SD: 7.0?±?9.63 pg/mL vs. 15.6?±?24.1 pg/mL, p = .013). Accordingly, the DLMO occurred at similar times in young (20:10?h) and older (19:57?h) good-sleeping women, but was delayed ～50?min in older poor-sleeping women (20:47?h). Older poor sleepers showed a shorter phase angle between DLMO and sleep onset, but a longer phase angle between CBT peak and sleep onset than young good sleepers, whereas older good sleepers had intermediate phase angles (insignificant). Regression analysis showed that the DLMO was a significant predictor of SOL in the older women (R²?=?0.64, p < .001), but not in the younger women. This indicates that melatonin production started later in those older women who needed more time to fall asleep. In conclusion, changes in melatonin level and CBT were intact in older poor sleepers in that evening melatonin increased and CBT decreased. However, poor sleepers showed a weaker evening increase in melatonin level, and their DLMO was delayed compared with good sleepers, suggesting that it is not primarily the absolute level of endogenous melatonin, but rather the timing of the circadian rhythm in evening melatonin secretion that might be related to disturbances in the sleep-wake cycle in older people. (Author correspondence: mdittmar@zoologie.uni-kiel.de)     

Clinical significance of analysis of the level of blood fat,CRP and hemorheological indicators in the diagnosis of elder coronary heart disease

Objective

To investigate the levels of blood fat, C-reactive protein (CRP) and hemorheological indicators in the elder patients with coronary heart disease (CHD), so as to provide evidence for prospective study and treatment of elder CHD.

Circadian rhythms of melatonin in haemolymph and optic lobes of Chinese mitten crab (Eriocheir sinensis) and Chinese grass shrimp (Palaemonetes sinensis)

This study is the first to examine the circadian rhythms of melatonin in Eriocheir sinensis and Palaemonetes sinensis, two economically important crustaceans. We collected haemolymph and optic lobes from both species every 4 h over a whole day cycle. Melatonin content was measured with high-performance liquid chromatography. E. sinensis haemolymph exhibited significant (p < 0.05) peaks in melatonin at 16:00 (0.180 ± 0.020 μg·mL^?1) and 24:00 (0.244 ± 0.055 μg·mL^?1), while eyestalks had significant peaks at 16:00 (72.377 ± 18.100 μg·eyestalk^?1) and 24:00 (98.756 ± 30.271 μg·eyestalk^?1). In P. sinensis, melatonin peaked significantly only at 16:00 in optic lobes (12.493 ± 1.475 μg·eyestalk^?1) (p < 0.05); no significant peaks were present in haemolymph. Thus, both E. sinensis and P. sinensis exhibit species-specific melatonin rhythms. Time of day should therefore be considered when examining the physiological status of both crustaceans, given the potential influence of fluctuating daily melatonin concentrations.     

ETB-mediated contraction differs between left descending coronary artery and its next branch

Pig left descending coronary artery (main artery) and its next branch (branch arteries) differ in many properties. Here we report on the receptor types and the Ca²⁺ pools utilized for endothelin (ET) contraction in 3 mm long de-endothelialized rings of the main (weight 7.38 ± 0.38 mg) and the branch (1.07 ± 0.03 mg) arteries. KCl (60 mM) contracted the main and the branch arteries with force of 41.8 ± 3.1 and 16.9 ± 1.0 mN (millinewton), respectively. Force of contraction for all the other agents was normalized taking the KCl value as 100%. We determined the total ET-induced responses using ET-1 and those mediated by ET_B using IRL1620. In Ca²⁺-containing solutions, ET-1 contracted the main arteries with pEC_B = 8.2 ± 0.1 and a maximum force of 98 ± 5%. The branch arteries also gave similar values of pEC₅₀ (8.4 ± 0.1) and maximum force (99 ± 14%). IRL1620 contracted the main and the branch arteries with pEC₅₀ = 7.9 ± 0.1 but the maximum force was significantly higher in the branch arteries (44 ± 3%) than in the main (15 ± 2%). In Ca²⁺-free solutions, the pEC₅₀ values for ET-1 or IRL-1620 did not change but the maximum force of contraction was diminished considerably in both main and branch arteries. Thus, the left coronary artery and its next branch differ in that the role of ET_B receptors is greater in the latter.     

Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study

HDL is strongly inversely related to cardiovascular risk. Hepatic HDL uptake is controlled by ecto-F₁-ATPase activity, and potentially inhibited by mitochondrial inhibitor factor 1 (IF1). We recently found that IF1 is present in serum and correlates with HDL-cholesterol (HDL-C). Here, we have evaluated the relationship between circulating IF1 and plasma lipoproteins, and we determined whether IF1 concentration is associated with the risk of coronary heart disease (CHD). Serum IF1 was measured in 648 coronary patients ages 45–74 and in 669 matched male controls, in the context of a cross-sectional study on CHD. Cardiovascular risk factors were documented for each participant, including life-style habits and biological and clinical markers. In controls, multivariate analysis demonstrated that IF1 was independently positively associated with HDL-C and apoA-I (r = 0.27 and 0.28, respectively, P < 0.001) and negatively with triglycerides (r = −0.23, P < 0.001). Mean IF1 concentration was lower in CHD patients than in controls (0.43 mg/l and 0.53 mg/l, respectively, P < 0.001). In multivariate analyses, following adjustments on cardiovascular risk factors or markers, IF1 was negatively related to CHD (P < 0.001). This relationship was maintained after adjustment for HDL-C or apoA-I. This study identifies IF1 as a new determinant of HDL-C that is inversely associated with CHD.     

Expression of bone morphogenetic protein 4 and its receptors in the remodeling heart

Aims

Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure.

Main methods

Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n = 15) or ischemic heart disease (CAD; n = 9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n = 7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n = 11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n = 7 at each time point).

Key findings

Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24 h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H₂O₂ injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia–reoxygenation injury.

Significance

Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes.     

Human Melatonin and Cortisol Circadian Rhythms in Patients with Coronary Heart Disease

Cortisol and melatonin have well known circadian rhythms, coupled to the solar day. Melatonin has been shown to serve as an endogenuous “Zeitgeber” (time giver) and is secreted by the human pineal gland throughout the night but not during the day. Patients with coronary heart disease (CHD) have significant depressed nocturnal melatonin secretion compared to healthy individuals (Brugger et al., 1995). In addition to our previous study we measured serum concentrations of cortisol to evaluate whether the circadian rhythm of cortisol secretion is also different in patients with CHD. Blood was collected by venous puncture at 0200 and at 1400, serum separated and kept frozen at -20°C until analysis. Cortisol and melatonin were measured with a commercially available radioimmunoassay according to the instructions of the manufacturer. Nineteen patients with angiographically documented CHD (mean age 53 years) participated in this study. As control group served 12 adults without any signs of CHD. Melatonin serum concentrations (median; mean ± SD) at night were significantly depressed in patients with coronary heart disease (7.8; 8.6 ± 3.3 pg/ml) in comparison to the control group (38.0; 45.4 ± 24.1 pg/ml) p &lt; 0.01. Melatonin in the afternoon was not detectable in either of the groups. Cortisol values at night were significantly raised in patients with coronary heart disease (6.0; 7.2 ± 3.7 µg/dl) in comparison to the control group (2.7; 3.8 ± 2.9 µg/dl) p &lt; 0.05. Cortisol levels in the afternoon were also elevated in patients with CHD (8.9; 9.5 ± 3.8 µg/dl) but there was no significant difference compared to controls (6.8; 6.9 ± 4.5 µg/dl). The results of the present study indicate that patients with coronary heart disease have atypical secretory patterns of nocturnal cortisol and melatonin secretion.     

Scaling with body mass of mitochondrial respiration from the white muscle of three phylogenetically, morphologically and behaviorally disparate teleost fishes

White muscle (WM) fibers in many fishes often increase in size from <50 μm in juveniles to >250 μm in adults. This leads to increases in intracellular diffusion distances that may impact the scaling with body mass of muscle metabolism. We have previously found similar negative scaling of aerobic capacity (mitochondrial volume density, V _mt) and the rate of an aerobic process (post-contractile phosphocreatine recovery) in fish WM. In the present study, we examined the scaling with body mass of oxygen consumption rates of isolated mitochondria (VO_2mt) from WM in three species from different families that vary in morphology and behavior: an active, pelagic species (bluefish, Pomatomus saltatrix), a relatively inactive demersal species (black sea bass, Centropristis striata), and a sedentary, benthic species (southern flounder, Paralichthys lethostigma). In contrast to our prior studies, the measurement of respiration in isolated mitochondria is not influenced by the diffusion of oxygen or metabolites. V _mt was measured in WM and in high-density isolates used for VO_2mt measurements. WM V _mt was significantly higher in the bluefish than in the other two species and VO_2mt was independent of body mass when expressed per milligram protein or per milliliter mitochondria. The size-independence of VO_2mt indicates that differences in WM aerobic function result from variation in V _mt and not to changes in VO_2mt. This is consistent with our prior work that indicated that while diffusion constraints influence mitochondrial distribution, the negative scaling of aerobic processes like post-contractile PCr recovery can largely be attributed to the body size dependence of V _mt.     

犬冠状动脉阻力的胆碱能调节

本实验在麻醉开胸犬,采用冠状动脉左旋支恒流灌注,于搏动的和心室纤颤(VF)的心脏,研究了电刺激迷走神经(VNS)及冠状动脉内注入乙酰胆碱(ACh)对冠状动脉阻力的影响。当 VNS 和冠脉内给 ACh 时,(1)心肌内小冠状动脉阻力显著减低,而心外膜大冠状动脉阻力并无明显变化;(2)冠状动脉左旋支总阻力的减低幅度在 VF 的心脏比在搏动的心脏显著减小。以上结果表明,迷走-ACh 扩张冠脉的作用主要是舒张心肌内小冠状动脉,并可通过减低心肌收缩力而间接降低冠状动脉阻力。     

Association of the melatonin circadian rhythms with clock 3111T/C gene polymorphism in Caucasian and Asian menopausal women with insomnia

A comparative analysis of melatonin circadian rhythms in Caucasian (incoming population) and Asian (indigenous population) menopausal women with/without sleep disorders depending on the genotype of Clock 3111T/C gene polymorphism was realized.The melatonin level in the saliva was determined four times a day (6:00–7:00, 12:00–13:00, 18:00–19:00, 23:00–00:00 h). The Caucasian women—carriers of the TT-genotype with insomnia as compared to control group—had a higher morning melatonin level and a lower night melatonin level. The Asian women with TT-genotype and insomnia had a lower levels of melatonin as compared to control at daytime, evening and night. A significantly higher melatonin level in the early morning hours was detected in the Caucasian women—carriers of the TT-genotype with insomnia as compared to group womencarriers of the minor 3111C-allele. There were no statistically significant differences in the circadian rhythms of melatonin in the Asian women depending on the genotype of the Clock 3111T/C polymorphism. An assumption with respect to the protective role of the minor allele 3111C in the development of insomnia associated with the displacement of melatonin circadian rhythms in the representatives of the incoming population was made.     

Screening of Ligand Binding on Melatonin Receptor Using Non-Peptide Combinatorial Libraries

Abstract

The screening of combinatorial libraries requires a deconvolution procedure to obtain, in fine, the most active compound of the starting library. the standard screening assays used in regular molecular pharmacology, have been poorly assessed when transposed to combinatorial chemistry-related experiments, particularly those involving large numbers of chemicals in a single assay. One key issue is the effect of the inactive analogs on the identification of the active ligand in mixtures. We chose melatonin receptors to measure the apparent affinity of a single ligand when tested alone or in mixtures of non-peptide low molecular weight compounds. Using ligands with IC₅₀ from the micro- to the picomolar range, mixed with increasingly complex mixtures of 5 to 20 or 25 inactive compounds, we analyzed the displacements from the mt₁ and MT₂ melatonin receptor subtypes of the radioligand 2-iodomelatonin (K_d= 25pmol/l and 200pmol/l, respectively). the behavior of equimolar mixtures in displacement curves led to the conclusion that the observed binding affinity reflects the dilution effect of mixing the active component with inactive compounds but does not reveal noticeable interactions which would interfere with the binding process. From the practical point of view, the concentrations of the active species in the binding assay should be large enough to displace significantly the radioligand, a requirement which may be limited by the solubility of the ligand mixtures. in contrast, previous observations with peptide libraries report that the dilution effect is often compensated by additive or synergic action of structurally related analogs, thus making possible the deconvolution of very large (typically up to 10⁷ compounds) peptide libraries.