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Abstract-A simple colorimetric assay using MTT has been developed to monitor mammalian cell survival and proliferation in vitro. In this study we used a clonal fibroblastic cell line (RPC-C2A) from rat incisal dental pulp to examine the effec tiveness of the colorimetric assay to test for the toxicity of eugenol, which is fre quently used to treat inflammed dental pulp. A technical problem encountered was the insolubility of MTT formazan, produced by the activity of mitochondria dehydrogenases.Dimethyl sulfoxide (DMSO) seemed to be the best solvent. Doses of eugenol causing a 50% inhibition in the colorimetric assay were calculated as 0.6 mM and 1 mM for cells in the growing phase and for cells at confluence, respectively.These values exist in the concentration range reported in the previous studies.Although the correlation between spectrophotometric absorbance and cell number was not completely linear, this method could be used effectively as a simple preliminary assay to test for the toxicity of dental drugs and materials.

BackgroundAtrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals.ObjectivesOur aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF.Methods and ResultsTrans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2±104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR Ca2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR　and α1-AR may play important roles in the development of AF.ConclusionsWe have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments.

Coronary angiographic and risk factor (RF) characteristics were analyzed in 133 Japanese patients with ischemic heart disease (IHD) who were less than 40 years old and who had undergone coronary angiography (CAG) during the past 10 years at six university hospitals in the Tokyo area. We compared the coronary angiographic characteristics of the subject group with those of 216 controls with coronary sclerosis detected by CAG who were more than 40 years old (older control group) and the RF characteristics with those of 133 sex- and age-matched volunteers (younger control group). Sixty seven percent of the subjects (89 cases) were diagnosed as having myocardial infarction (MI) and 33% (44 cases) had angina pectoris (AP). Coronary artery disorders in this group consisted of 103 (77%) cases of coronary sclerosis, 20 (15%) cases of coronary spasm and 10 (8%) cases of miscellaneous diseases, eg, possible vasculitis with connective tissue disease, congenital anomalies, etc. The incidences of significant (> or = 75%) sclerotic narrowing in 0 vessels (31%) and 1 vessel (49%) in the subject group were significantly (p < 0.01) higher than those in the older control group, while the incidence of multivessel disease was significantly (p < 0.05) less in the subject group than in the older control group. The incidences of the following coronary risk factors were significantly (p < 0.05) higher in the subjects than in the younger controls: smoking (83% vs 35%), hypercholesteremia (44% vs 10%), obesity (31% vs 9%), hypertension (29% vs 3%), familial IHD (28% vs 7%) and diabetes mellitus (19% vs 2%). Thus, zero- or single-vessel disease predominated in the younger subject group and the prevalence of coronary risk factors was significantly higher in the subject.

The cytotoxicities of various phenolic compounds were examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay and established rat dental pulp cells (RPC-C2A). The ID50 (50% inhibition concentration) of the phenolic compounds ranged from 0.1 to 10 mmol/L, and the cytotoxicities of the phenolic compounds were related to their phenolic coefficients. The therapeutic index of each phenolic compound was calculated from the ID50 results and from the 50% inhibition concentration for prostaglandin synthesis that has been reported previously. Eugenol, guaiacol, and creosol had therapeutic indices much higher than those of other phenolic compounds examined. This experimental system could be used as a simple preliminary assay to test for toxicity of dental drugs and materials.

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.

In the present study, an attempt was made to clarify whether ANP molecular forms in the plasma of severe congestive heart failure patients differ from those in healthy persons and whether ANP molecular forms in the plasma of the patients were changed by successful treatment of cardiac disease. Twenty patients with congestive heart failure were treated at Kitasato University Hospital. They were classified as class III or IV by New York Heart Association criteria at the time of admission. Plasma ANP concentrations decreased after treatment from 356 +/- 58.2 to 72.3 +/- 14.8 pg/ml. The gel permeation chromatograms from the plasma of healthy persons showed low, or low and high molecular weight ANP peaks which correspond to the elution positions of authentic alpha-ANP or ribonuclease A (mol. wt., 13.7 kdalton). In patients with severe congestive heart failure at a severe stage, middle molecular weight ANP consisted with the elution position of authentic beta-ANP was particularly noted in addition of low and high molecular weight ANP peaks. This middle molecular weight peak disappears in most of cases by successful treatment. Molecular forms in the plasma obtained from the coronary sinus and the inferior or superior vena cava were essentially the same. These results indicate that the middle molecular weight ANP supposed as beta-ANP may particularly be secreted in severe congestive heart failure patients.

The C=C stretching frequencies of N,N'-derivatives of indigo reveal that although these compounds, with the exception of N,N-oxalylindigo, are considerably twisted about the central C=C bond owing to steric repulsion, the deviation from the coplanarity of the two halves of the molecule is not very different between the trans and cis isomers: the cis isomers are not always more twisted than the corresponding trans isomers. In contrast to the commonly accepted interpretation, the large hypsochromic shifts brought about by the trans + cis isomerizatioo of N,N'derivatives of indigo cannot be explained as being due to a departure from coplanarity in the cis con&-uration.