Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis

Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-alpha-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-alpha-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.     

Leptin and post-prandial satiety: acute central leptin more potently reduces meal frequency than meal size in the rat

Rationale Many attempts to understand ingestion have sought to clarify the control of meals. Little is known about the effects of the anorexogenic hormone leptin on meal patterning.Objective The present study sought to perform a dose–response analysis of the effects of acute central leptin administration on meal patterning using a validated, objective meal definition and to compare these results to those obtained with a previously used, subjective meal definition.Methods To validate the objective meal definition pharmacologically, the microstructural effects of the well-studied compound fenfluramine (SC 0, 1, 2, 4 mg/kg) on spontaneous nocturnal intake were determined in mature, non-deprived male Wistar rats (n=8) using a full Latin square design. The effects of intracerebroventricular leptin administration (0, 0.3, 1, 3, 6.25 g; n=10) were also examined, and perceived meal patterns obtained from the objective and subjective definitions were compared.Results Fenfluramine reduced meal size and eating rate at doses that did not reduce meal frequency or duration. In contrast, comparably anorectic doses of leptin had potent post-meal satiety-like effects, reducing meal frequency and prolonging the intermeal interval without reducing average meal size, a finding opposite to that suggested by the previously used subjective meal definition. Unlike comparably and more anorectic doses of fenfluramine, leptin non-specifically reduced both prandial and non-prandial drinking.Conclusions Acute increases in central leptin levels may potently augment post-prandial satiety and influence body-fluid homeostasis. The results reveal unappreciated central modes of action for the ob protein which qualitatively differ from the intra-meal satiating-like effects of fenfluramine.     

Unique excitation–contraction characteristics of mouse myocardium as revealed by SEA0400, a specific inhibitor of Na<Superscript>+</Superscript>–Ca<Superscript>2+</Superscript> exchanger

The functional role of the sodium–calcium exchanger in mouse ventricular myocardium was evaluated with a newly developed specific inhibitor, SEA0400. Contractile force and action potential configuration were measured in isolated ventricular tissue preparations, and cell shortening and Ca²⁺ transients were measured in indo-1-loaded isolated ventricular cardiomyocytes. SEA0400 increased the contractile force, cell shortening and Ca²⁺ transient amplitude, and shortened the late plateau phase of the action potential. -adrenergic stimulation by phenylephrine produced a sustained decrease in contractile force, cell shortening and Ca²⁺ transient amplitude, which were all inhibited by SEA0400. Increasing the contraction frequency resulted in a decrease in contractile force in the absence of drugs (negative staircase phenomenon). This frequency-dependent decrease was attenuated by SEA0400 and enhanced by phenylephrine. Phenylephrine increased the Ca²⁺ sensitivity of contractile proteins in isolated ventricular cardiomyocytes, while SEA0400 had no effect. These results provide the first pharmacological evidence in the mouse ventricular myocardium that inward current generated by Ca²⁺ extrusion through the sodium–calcium exchanger during the Ca²⁺ transient contributes to the action potential late plateau, that -adrenoceptor-mediated negative inotropy is produced by enhanced Ca²⁺ extrusion through the sodium–calcium exchanger, and that the negative staircase phenomenon can be explained by increased Ca²⁺ extrusion through the sodium–calcium exchanger at higher contraction frequencies.     

Allergic airway eosinophilia is suppressed in ovalbumin-sensitized Brown Norway rats fed raffinose and alpha-linked galactooligosaccharide

We recently found that dietary raffinose suppressed allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats. Using this model in the present study, we compared the efficacy of other oligosaccharides with that of raffinose. Brown Norway rats were immunized s.c. with ovalbumin on d 0 and exposed to aerosolized ovalbumin on d 20; broncho-alveolar lavage fluid was obtained on d 21. In Expt. 1, rats were fed a control diet or diets supplemented with different oligosaccharides (50 g/kg diet, raffinose, alpha-linked galactooligosaccharide, fructooligosaccharide, and xylooligosaccharide). The number of eosinophils in the fluid was significantly lower in rats fed raffinose and alpha-linked galactooligosaccharide diets than in those fed the control diet. Dietary fructooligosaccharide and xylooligosaccharide did not affect airway eosinophilia. In Expt. 2, i.p. administration of raffinose and alpha-linked galactooligosaccharide, but not fructooligosaccharide and xylooligosaccharide, suppressed airway eosinophilia in rats fed the control diet. In Expt. 3, suppression of airway eosinophilia by dietary alpha-linked galactooligosaccharide occurred in cecectomized rats administered neomycin. Reduced levels of interleukin (IL)-4 and IL-5 mRNA in lung tissue were associated with the suppression of airway eosinophilia. We propose that indigestible oligosaccharides differ in their suppressive effect on allergic airway eosinophilia in ovalbumin-sensitized Brown Norway rats and that the effect appears not to be mediated by intestinal microflora.     

Resistance to diet-induced obesity in mu-opioid receptor-deficient mice: evidence for a "thrifty gene"

Using pharmacological tools, a role for opioid receptors in the regulation of food intake has been documented. However, the involvement of specific receptor subtypes remains questionable, and little information is available regarding a role for opioid receptors in energy metabolism. Using adult male mice lacking the mu-opioid receptor (MOR) gene (MOR-/-), we show that the MOR is not essential for the maintenance of normal levels of ad libitum food intake but does modulate the efficiency of energy storage during high-fat diets through the regulation of energy partitioning. When fed a regular diet, MOR-/- mice displayed only subtle alterations in energy homeostasis, suggesting a relative overuse of fat as a fuel source in the fed state. When fed a high-fat diet, MOR-/- mice were resistant to obesity and impaired glucose tolerance, despite having similar energy intake to wild-type mice. This resistance to obesity was associated with a strong induction of the expression of key mitochondrial enzymes involved in fatty acid oxidation within skeletal muscle. This metabolic role of the MOR, which is consistent with the properties of a "thrifty gene," suggests that the MOR pathway is a potential target for pharmacological intervention in the treatment of obesity associated with the intake of fatty diets.     

Intracranial hypertension in a boy with Mycoplasma pneumoniae infection: the first report

We describe a 7-y-old boy who developed idiopathic intracranial hypertension (IIH) during the convalescent phase of Mycoplasma pneumoniae (M. pneumoniae) infection. Anti-M. pneumoniae antibody and cold hemagglutinin titer were extremely high during his course, and corticosteroid administration was noticeably effective. It supports an immunopathological mechanism for his intracranial hypertension.     

The clinical outcome and quality of life following minimally invasive direct coronary artery bypass surgery

BACKGROUND: Minimally invasive direct coronary artery bypass (MIDCAB) through a limited anterior small thoracotomy has been shown to be a promising technique of surgical treatment for single or double vessel disease. Little is known about the Health-Related Quality Of Life (HRQOL) in this group of patients. METHODS: The records of 75 consecutive patients who underwent MIDCAB procedure at Harefield Hospital between April 2000 and January 2002 were reviewed retrospectively. HRQOL assessment was planned in a cross-sectional design. Patients were contacted by telephone to conduct a semi-structured interview and were sent two questionnaires: the Short Form health survey (SF-36) and the Hospital Anxiety and Depression Scale (HADS). RESULTS: There was no in-hospital death. Patients stayed in the Intensive Therapy Unit (ITU) for 11.56 +/- 4.55 hours and stayed in hospital for 3 +/- 2.34 days. None of the study patients had perioperative myocardial infarction (MI) or neurological complications including permanent and transient strokes. We were able to contact all the 75 patients by telephone and they also completed the SF-36 and HADS. The SF-36 scores were compared to an age-matched group of normal British people. The MIDCAB group had an excellent general health perception compared to the normal group (p < 0.001), but similar scores otherwise. The HADS scores showed that only 1 patient (1.3%) had mild depression, 5 patients (6.7%) had mild anxiety, and 2 patients (2.6%) had moderate anxiety. CONCLUSION: MIDCAB is a safe surgical treatment and provides excellent clinical and HRQOL outcomes.