The function of animal ‘eyespots’:Conspicuousness but not eye mimicry is key

Many animals are marked with conspicuous circular features often called 'eyespots',which intimidate predators,preventing or halting an attack.It has long been assumed that eyespots work by mimicking the eyes of larger animals,but recent experiments have indicated that conspicuousness and contrast is important in eyespot function,and not eye mimicry.We undertake two further experiments to distinguish between the conspicuousness and mimicry hypotheses,by using artificial prey presented to wild avian predators...     

Mitochondrial,but not peroxisomal, β-oxidation of fatty acids is conserved in coenzyme A-Deficient rat liver

Growth hormone (GH) exerts acute insulin-like effects, such as increased lipogenesis and inhibition of catecholamine-induced lipolysis, in rat adipocytes that have not been exposed to GH during the preceding three hours. We found that OPC3911, a highly specific inhibitor of the cGMP-inhibited cAMP phosphodiesterase, completely blocked the antilipolytic but not the lipogenic effect of GH. This indicates that the antilipolytic effect of GH is mediated through activation of this phosphodiesterase leading to reduction of cAMP levels in the same manner as has been shown for insulin.     

The effect of body temperature on the dynamic respiratory system compliance–breathing frequency relationship in the rat

The mechanical inhomogeneity of the respiratory system is frequently investigated by measuring the frequency dependence of dynamic compliance, but no data are currently available describing the effects of body temperature variations. The aim of the present report was to study those effects in vivo. Peak airway pressure was measured during positive pressure ventilation in eight anesthetized rats while breathing frequency (but not tidal volume) was altered. Dynamic compliance was calculated as the tidal volume/peak airway pressure, and measurements were taken in basal conditions (mean rectal temperature 37.3 °C) as well as after total body warming (mean rectal temperature 39.7 °C). Due to parenchymal mechanical inhomogeneity and stress relaxation-linked effects, the normal rat respiratory system exhibited frequency dependence of dynamic lung compliance. Even moderate body temperature increments significantly reduced the decrements in dynamic compliance linked to breathing rate increments. The results were analyzed using Student’s and Wilcoxon’s tests, which yielded the same results (p < 0.05). Body temperature variations are known to influence respiratory mechanics. The frequency dependence of dynamic compliance was found, in the experiments described, to be temperature-dependent as temperature variations affected parenchymal mechanical inhomogeneity and stress relaxation. These results suggest that body temperature variations should be taken into consideration when the dynamic compliance–breathing frequency relationship is being examined during clinical assessment of inhomogeneity of lung parenchyma in patients.     

Electromyographic,but not mechanomyographic amplitude–force relationships,distinguished differences in voluntary activation capabilities between individuals

The purpose of the present study was to examine the influence of activation capabilities on the electromyography (EMG_RMS) and mechanomyography amplitude (MMG_RMS)–force relationships of the vastus lateralis (VL) and rectus femoris (RF). Thirteen men (mean ± SD; age = 22 ± 3 year) performed nine submaximal contractions (10–90% maximal voluntary contraction [MVC]) with the interpolated twitch technique performed during a separate contraction at 90% MVC to calculate percent voluntary activation (%VA). Nine participants with >90% VA were categorized into the high-activated group with the remaining categorized into the moderate-activated group. Slopes (b terms) were calculated from the log-transformed EMG_RMS and MMG_RMS–force relationships. The b terms (collapsed across the VL and RF) for the EMG_RMS–force relationships were greater for the high- (1.29 ± 0.31) than the moderate-activated (1.10 ± 0.20) group. In contrast, there were no differences in the b terms for the MMG_RMS–force relationships between the high- and moderate-activated groups. For the EMG_RMS and MMG_RMS–force relationships, the b terms were greater for the RF (1.38 ± 0.30, 0.81 ± 0.20) than the VL (1.08 ± 0.19, 0.60 ± 0.13) collapsed across groups. The b terms from the EMG_RMS–force relationships, but not the MMG_RMS–force relationships, reflected differences in %VA.     

Differences in the force–interval relationship of isolated human myocardium with chronic coronary artery disease with and without type 2 diabetes mellitus and the role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPase

The dynamics of the force–interval relationship of the human myocardium in coronary artery disease (CAD) and CAD with concomitant diabetes mellitus was studied, and its dependence on the level of sarcoplasmic reticulum (SR) Ca²⁺-ATPase expression was evaluated. The study was performed on myocardial biopsy material obtained during coronary bypass operation using cardiopulmonary techniques. Patients with chronic CAD and patients with CAD associated with type II diabetes mellitus were enrolled in the study. It was found that CAD patients with and without diabetes mellitus with similar clinical parameters had either negative or positive dynamics of the force–interval relationship. The positive force–interval relationship was associated with a “high level” of Ca²⁺-ATPase, while the negative force–interval relationship was associated with a “low level” of this protein. In CAD associated with diabetes mellitus of short duration, the positive dynamics of the force–interval relationship is more pronounced and corresponds to a higher level of SR Ca²⁺-ATPase expression than in CAD alone.     

The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Serine O-acetyltransferase is important,but not essential for cysteine–methionine synthesis in Fusarium graminearum

O-acetyltransferase (SAT) is a key enzyme converting serine into O-acetylserine in the synthesis of sulphur-containing amino acids. To characterize the function of FgSAT in Fusarium graminearum, three deletion mutants of FgSAT (ΔFgSAT-1, -2 and -18) were obtained using a gene replacement strategy. The three mutants did not show recognizable phenotypic changes on potato dextrose agar medium, but exhibited a very weak growth on fructose gelatin agar (FGA) medium containing SO₄ ^2? as sole sulfur source. Supplementation of O-acetylserine, cysteine, or methionine, but not serine, rescued the defect of mycelial growth in FgSAT deletion mutants, indicating that FgSAT is involved in conversion of serine into O-acetylserine. The three mutants had a decrease in conidiation in mung bean liquid, but not in carboxymethyl cellulose. Virulence, deoxynivalenol production and fungicide sensitivity assays found that the three mutants showed no significant difference from wild-type progenitor PH-1. Real-time PCR assays detected an increase in expression levels of FgOAHS, FgCBS and FgCGL genes involved in the alternative pathway in FgSAT deletion mutants, suggesting that the alternative pathway in F. graminearum is present and can operate. Addition of homoserine, the upstream substrate of the alternative pathway, also restored the normal mycelial growth of FgSAT deletion mutants on FGA, indicating that the alternative pathway in F. graminearum might be positively regulated by homoserine.     

The lunar cycle,sunspots and the frequency of births in Germany, 1920–1989

Based on multivariate linear regression models, we analyze the effect of the lunar cycle and the number of sunspots occurring on a particular day on the number of births using social security data and controlling for a number of other potential confounders. The daily numbers of births between 1920 and 1989 have been calculated from the full sample of individuals who have been registered at least once in the German social security system. While the lunar cycle does not affect the number of births, the number of sunspots is positively correlated to the number of births. The empirical results may be explained by medical technological progress making natural influences on births less important over time. This interpretation is supported by the results on the intertemporal influence of weekends and holidays on the frequency of daily births.     

The l-α-amino acid receptor GPRC6A is expressed in the islets of Langerhans but is not involved in l-arginine-induced insulin release

GPRC6A is a seven-transmembrane receptor activated by a wide range of l-α-amino acids, most potently by l-arginine and other basic amino acids. The receptor is broadly expressed, but its exact physiological role remains to be elucidated. It is well established that l-arginine stimulates insulin secretion; therefore, the receptor has been hypothesized to have a role in regulating glucose metabolism. In this study, we demonstrate that GPRC6A is expressed in islets of Langerhans, but activation of the receptor by l-arginine did not stimulate insulin secretion. We also investigated central metabolic parameters in GPRC6A knockout mice compared with wildtype littermates and found no difference in glucose metabolism or body fat percentage when mice were administered a standard chow diet. In conclusion, our data do not support a role for GPRC6A in l-arginine-induced insulin release and glucose metabolism under normal physiological conditions.     

The ��-subunit of ATP synthase is involved in cellular uptake and resecretion of apoA-I but does not control apoA-I-induced lipid efflux in adipocytes

Cellular uptake and resecretion of apoA-I (apoA-I recycling) could be an important factor in determining the circulating plasma levels of apoA-I and/or HDL. Using a novel method to study protein recycling, we have recently demonstrated recycling of apoA-I by adipocytes and suggested that this is a receptor mediated process independent of ABCA1 function. In the present study, it is shown that apoA-I recycling by adipocytes can be blocked by a monoclonal antibody against the β-subunit of ATP synthase, a protein that had been previously identified as an apoA-I receptor. Investigation of the cellular recycling of two other proteins, an apolipoprotein and a small globular protein, showed that recycling of apoA-I is a selective process. The present study also shows that blocking apoA-I recycling has no effect on the rate of apoA-I-induced cholesterol or phospholipid efflux. It is concluded that cellular recycling of apoA-I is a selective process that involves the ectopically expressed β-subunit of ATP synthase. The physiological function of apoA-I recycling remains to be elucidated. However, this study shows that the process of apoA-I uptake and resecretion is not required for apoA-I lipidation.     

Treatment with cholesterol-loaded methyl-β-cyclodextrin increased the cholesterol in rabbit oocytes,but did not improve developmental competence of cryopreserved oocytes

Membrane cholesterol:phospholipids ratio is an important determinant of cell chilling sensitivity. At low temperatures, major membrane destabilisation occurs when the membrane undergoes a phase transition. To increase membrane fluidity and stability during cooling and thus increase oocyte cryoresistance, cholesterol has been added to the plasma membrane. This study was conducted to determine if cholesterol could be incorporated into rabbit oocytes by incubation with cholesterol-loaded methyl-β-cyclodextrin (CLC) and if added cholesterol could improve the developmental ability of cryopreserved oocytes after parthenogenetic activation or intracytoplasmic sperm injection. Fresh, frozen and vitrified oocytes incubated with CLC containing 20% NBD-labelled cholesterol (NBD-CLC) were evaluated using confocal microscopy. Fluorescence intensity was higher in fresh oocytes than in cryopreserved ones. Pre-treating rabbit oocytes with 1 mg of NBD-CLC/mL did not improve cleavage and developmental rates after cryopreservation. Results showed that treatment with CLC increased the cytoplasmic cholesterol content, but did not improve cleavage rate and developmental competence of cryopreserved oocytes.     

The increase in rat ventricular automaticity induced by salbutamol is mediated through β(1)- but not β(2)-adrenoceptors: role of phosphodiesterases

AimsWhile β₂-adrenoceptor (AR) agonists are useful bronchodilators, they also produce cardiac arrhythmias. These agents are not fully selective and also activate β₁-AR, but the involvement of β₁-AR and β₂-AR in the observed pro-arrhythmic effect has not been established. We studied the effect of β₁-AR and β₂-AR activation on ventricular automaticity and the role of phosphodiesterases (PDE) in regulating this effect.Main methodsExperiments were performed in the spontaneously beating isolated right ventricle of the rat heart. We also measured cAMP production in this tissue.Key findingsThe β₂-AR agonist salbutamol (1-100 μM) produced a concentration-dependent increase in ventricular automaticity that was not affected by 50 nM of the β₂-AR antagonist ICI 118551. This effect was enhanced by the non-selective PDE inhibitor theophylline (100 μM) and by the selective PDE4 inhibitors rolipram (1 μM) and Ro 201724 (2 μM), but not modified by the selective PDE3 inhibitors cilostamide (0.3 μM) or milrinone (0.2 μM). The effects of salbutamol alone and in the presence of either theophylline or rolipram were virtually abolished by 0.1 μM β₁-AR antagonist CGP20712A. Salbutamol (10 μM) increased the cAMP concentration, and this effect was abolished by CGP 20712A (0.1 μM) but enhanced by theophylline (100 μM) or rolipram (1 μM). Cilostamide (0.3 μM) failed to modify the effect of salbutamol on cAMP concentration.SignificanceThese results indicate that the increase of ventricular automaticity elicited by salbutamol was exclusively mediated through β₁-AR and enhanced by non-selective PDE inhibition with theophylline or selective PDE4 inhibition. However, PDE3 did not appear to regulate this effect.     

Localization of α1,2,3-subunit isoforms of Na,K-ATPase in cultured neonatal and adult rat myocardium: The immunofluorescence and immunocytochemical study

By indirect immunofluorescence and preembedding peroxidase-diaminobenzidine technique the localization of polyclonal and monoclonal antibodies against 1, 2 and 3 isoforms of the Na,K-ATPase were studied in rat myocardium.The 1-subunit was identified predominantly on sarcolemma of cultured myocytes, neonatal, as well as adult cardiocytes. The 2 signal was localized around nuclei of cultured cardiocytes, very weak signals were seen in neonatal and more intense signal, were dispersed throughout the adult myocytes. The 3-subunit immunoreactivity was weak and localized in cell processes connecting individual cultured cells, on sarcolemma and intercalated discs of neonatal cells and very weak in adult working myocytes. Cytochemically demonstrated ouabain resistant Na,K-ATPase localized in junctional sarcoplasmic reticulum may represent 1 isoenzyme which is directly involved in modulation of action potential fluxes.     

Undermethylation at the 5′ end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture

We have measured methylation of the albumin gene in clones of rat hepatoma cells that vary quantitatively in their rates of synthesis of albumin and in variant and hybrid cells that produce no albumin. Although the albumin gene is undermethylated for its entire length in rat liver, only the 5′ end is ever undermethylated in hepatoma cells. Moreover, undermethylation of the 5′ end of the gene appears to be necessary for stable expression of the albumin gene in hepatoma cells. Since undermethylation of this region is found in some variant cells that fail to produce albumin, it is not a sufficient condition for albumin gene expression. Despite the excellent correlation between undermethylation of the 5′ end of the albumin gene and its stable expression, the results argue against the possibility that the methylated state of such genes during development determines whether they will or will not be expressed.     

Central infusion of leptin improves insulin resistance and suppresses β-cell function,but not β-cell mass,primarily through the sympathetic nervous system in a type 2 diabetic rat model

AimsWe investigated whether hypothalamic leptin alters β-cell function and mass directly via the sympathetic nervous system (SNS) or indirectly as the result of altered insulin resistant states.Main methodsThe 90% pancreatectomized male Sprague Dawley rats had sympathectomy into the pancreas by applying phenol into the descending aorta (SNSX) or its sham operation (Sham). Each group was divided into two sections, receiving either leptin at 300 ng/kg bw/h or artificial cerebrospinal fluid (aCSF) via intracerebroventricular (ICV) infusion for 3 h as a short-term study. After finishing the infusion study, ICV leptin (3 μg/kg bw/day) or ICV aCSF (control) was infused in rats fed 30 energy % fat diets by osmotic pump for 4 weeks. At the end of the long-term study, glucose-stimulated insulin secretion and islet morphometry were analyzed.Key findingsAcute ICV leptin administration in Sham rats, but not in SNSX rats, suppressed the first- and second-phase insulin secretion at hyperglycemic clamp by about 48% compared to the control. Regardless of SNSX, the 4-week administration of ICV leptin improved glucose tolerance during oral glucose tolerance tests and insulin sensitivity at hyperglycemic clamp, compared to the control, while it suppressed second-phase insulin secretion in Sham rats but not in SNSX rats. However, the pancreatic β-cell area and mass were not affected by leptin and SNSX, though ICV leptin decreased individual β-cell size and concomitantly increased β-cell apoptosis in Sham rats.SignificanceLeptin directly decreases insulin secretion capacity mainly through the activation of SNS without modulating pancreatic β-cell mass.     

Calmodulin·(Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM · (Ca²⁺)₄ is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01–1 μ M) was 0.9 nM; the respective apparent dissoclation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1–50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by ¹²⁵I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM · (Ca²⁺)₄. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E · CaM) is at least 100-fold greater than the apparent dissociation constant of the E · CaM · (Ca²⁺)₄ complex, as judged from non-saturation ¹²⁵I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.     

The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.     

The flavonoid luteolin inhibits Fcγ-dependent respiratory burst in granulocytes, but not skin blistering in a new model of pemphigoid in adult mice

Bullous pemphigoid is an autoimmune blistering skin disease associated with autoantibodies against the dermal-epidermal junction. Passive transfer of antibodies against BP180/collagen (C) XVII, a major hemidesmosomal pemphigoid antigen, into neonatal mice results in dermal-epidermal separation upon applying gentle pressure to their skin, but not in spontaneous skin blistering. In addition, this neonatal mouse model precludes treatment and observation of diseased animals beyond 2–3 days. Therefore, in the present study we have developed a new disease model in mice reproducing the spontaneous blistering and the chronic course characteristic of the human condition. Adult mice were pre-immunized with rabbit IgG followed by injection of BP180/CXVII rabbit IgG. Mice pre-immunized against rabbit IgG and injected 6 times every second day with the BP180/CXVII-specific antibodies (n = 35) developed spontaneous sustained blistering of the skin, while mice pre-immunized and then treated with normal rabbit IgG (n = 5) did not. Blistering was associated with IgG and complement C3 deposits at the epidermal basement membrane and recruitment of inflammatory cells, and was partly dependent on Ly-6G-positive cells. We further used this new experimental model to investigate the therapeutic potential of luteolin, a plant flavonoid with potent anti-inflammatory and anti-oxidative properties and good safety profile, in experimental BP. Luteolin inhibited the Fcγ-dependent respiratory burst in immune complex-stimulated granulocytes and the autoantibody-induced dermal-epidermal separation in skin cryosections, but was not effective in suppressing the skin blistering in vivo. These studies establish a robust animal model that will be a useful tool for dissecting the mechanisms of blister formation and will facilitate the development of more effective therapeutic strategies for managing pemphigoid diseases.