Isoamylamine Induces B16-F1 Melanoma Cell Autophagy by Upregulating the 5' Adenosine Monophosphate-Activated Protein Pathway.

Isoamylamine (IA) is an aliphatic monoamine molecule present in cheese, eggs, and wine. It belongs to the family of polyamines and also can be synthesized endogenously. It has been known that regulation of polyamines in cells is related to cell cycle and tumor formation. Malignant melanoma is difficult to treat and easily resistant to chemotherapy/radiotherapy through autophagy. In this study, we aim to clarify whether IA has a growth control effect on melanoma tumor cells and the regulatory mechanism. We treated B16-F1 melanoma cells with IA at concentrations of 0, 200, 400, and 600 ppm for 24 h. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was checked for cell viability and results showed that IA has an inhibitory effect on B16-F1 melanoma cells. The signaling molecules, which included Raf/MEK/ERK, were activated, while MSK1 and protein kinase B (AKT) were suppressed. Autophagy was also confirmed to be induced by IA. The acridine orange stain-positive cells were increased and BECN-1/LC3 upregulated. The data also showed that the autophagy regulatory molecule, 5'-adenosine monophosphate-activated protein kinase (AMPK), was induced after IA treatment, so we used dorsomorphin to inhibit AMPK and found that it could suppress autophagy. In conclusion, IA has an effect of inducing autophagy in B16-F1 cells and it is regulated through AMPK.

Novel Function of Isoamylamine Improves Survival in Endotoxemic Mice by Ameliorating Coagulopathy and Attenuating MMP-9 Expression Through p-ERK/p-p38 Signaling at Early Stage.

When a host suffers endotoxemic shock or septic shock, it results in many symptoms including disseminated intravascular coagulation (DIC). Septic shock (SS) causes coagulation time to decrease and then gradually increase, finally becoming prolonged and giving rise to DIC. Isoamylamine (IA) is one of the main components of grape products and can improve the survival rate of endotoxin lipopolysaccharide (LPS)-induced endotoxemic shock. The aim of this study was to elucidate if IA ameliorates coagulopathy in the early phase of LPS-induced damage. We studied the effects of IA on the coagulation system of extrinsic (prothrombin time [PT]) and intrinsic (activated partial thromboplastin time [aPTT]) pathways. PT and aPTT were tested in plasma drawn from mice following intraperitoneal (IP) injection of 1 mL of 1,000 ppm IA after LPS administration. Shortened PT was ameliorated by 1,000 ppm IA 1 h after LPS administration, but there was no effect on aPTT. In conclusion, IA 1,000 ppm partially intervenes in the early phase of LPS-induced damage, shortening plasma PT 1 h after LPS treatment in mice. Furthermore, we found 1,000 ppm IA also could attenuate MMP-9 expression through p-ERK/p-p38 signaling in mice hepatocyte extracts. This study focused on the effects of IA on blood coagulation function and inflammatory proteins. In the current situation of absence of effective treatment for SS, IA can increase survival rate and may offer another choice of patient avoiding causing death during endotoxemic shock.

The Anthropometric Measurement of Schober's Test in Normal Taiwanese Population.

The measurement of lower back mobility is essential in the assessment of lower back pain including ankylosing spondylitis. Original Schober's test (OST) and modified Schober's test (MST) are popularly conducted in daily rheumatology and orthopedics clinical practices. To our knowledge, this report is the only anthropometric reference study in a normal oriental population. The OST declined with age from 5.0 cm in the youngest (20-30 years old) to 3.1 cm in the aged (70-80 years old) male subjects and from 3.6 cm to 2.4 cm in the female subjects. The male OST was significantly more than the female OST. There was a good correlation between OST and MST in each of the three age groups of both sexes.

Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation.

PURPOSE: Ultraviolet B (UVB) irradiation activates nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in the cornea, resulting in inflammatory responses and malondialdehyde (MDA) accumulation. This study aims to determine the effect of zerumbone, a potent NF-κB inhibitor and inflammation modulators, on UVB-induced corneal damages in a mouse model. METHODS: Fifty female imprinting control region (ICR) mice were randomly divided into five groups. The mice were anaesthetized with their ocular surfaces exposed to UVB light (0.72J/cm(2)/daily), followed by daily dietary zerumbone supplements at 0, 1, 10, and 100 mg/kg of bodyweight. Mice without zerumbone supplements were used as treatment controls and mice without UVB irradiation as blank controls. Corneal surface damages were graded according to smoothness, opacity, and the extent of lissamine green staining. Histopathological changes were also examined, along with the expression of NF-κB, iNOS, and tumor necrosis factor-α (TNF-α). MDA accumulation and the levels of two antioxidant enzymes, glutathione (GSH) and GSH reductase (GR) were also examined. RESULTS: UVB irradiation caused significant damages to cornea, including sustained inflammation, apparent corneal ulcer, and severe epithelial exfoliation, leading to thinning of corneal epithelial layer, and infiltration of polymorphonuclear leukocytes. NF-κB expression was highly activated with nuclear translocation. The expression of iNOS and TNF-α were increased. MDA accumulation was also increased in both the corneal epithelial layer and the stroma. With dietary zerumbone, corneal damages were ameliorated in a dose-dependent manner. NF-κB activation and its nuclear translocation were blocked with decreased expression of iNOS and TNF-α. Infiltration of polymorphonuclear leukocytes was also blocked by dietary zerumbone. Besides, MDA accumulation was reduced with concomitant increase of GSH and GR levels. CONCLUSIONS: Dietary zerumbone prevents UVB-induced corneal damages by inhibition of NF-κB, iNOS, and TNF-α, with concomitant reduction of MDA accumulation and increase of GSH and GR levels in the mouse model. Results of this study suggest that dietary zerumbone may be used as a prophylactic agent against UVB-induced photokeratitis.

Dietary zerumbone prevents against ultraviolet B-induced cataractogenesis in the mouse.

PURPOSE: To investigate the preventive effect of dietary zerumbone against UVB-induced cataractogenesis. METHODS: A total of 50 six-week-old female ICR mice were split into five groups (each contained 10 mice) and exposed to UVB (0.72 J/cm(2)/daily) at noon for 7 days, except for the blank control group. The mice with UVB exposure were fed with zerumbone as a dietary supplement at 0, 1, 10, and 100 mg/kg of bodyweight, respectively, starting from one day before UVB exposure. On day 7, at 4 h after UVB exposure, all mice were subjected to cataract examination and lens opacity scoring, in correlation with levels of MDA (malondialdehyde), GSH (glutathione), GR (GSH reductase), GPx (glutathione peroxidase), and SOD (superoxide dismutase) in the lens. RESULTS: Dietary zerumbone at 100 mg/kg after UVB exposure was effective in decreasing lens opacity scores (p<0.001) and to reduce MDA (p<0.001), while GSH and GR levels were significantly increased (both p<0.001) in the lens. SOD was also increased with dietary zerumbone at 100 mg/kg (p=0.115), whereas GPx (p=0.171) levels were lower as compared with those without zerumbone after UVB exposure. CONCLUSIONS: These results suggest that zerumbone may protect against UVB-induced cataractogensis through reducing lipid peroxides and enhancing the endogenous antioxidant GSH level and GR activity.

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the ß-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

The significance of arginase I administration on the survival of mice bearing NS-1 myeloma cells.

BACKGROUND: Arginase I blood levels elevate in cancerous patients and correlate with cancer stages and poor prognosis. Since arginase is capable of enhancing cell growth, it is unclear whether its ominous effect on cancer progression is through the inhibition of immunity or through direct enhancement of cancer cell growth. We tried to clarify this question. METHODS: NS-1 mouse myeloma cells were inoculated intraperitoneally (i.p.) into mice. Purified mouse arginase I was injected daily either intravenously (i.v.) or i.p. for 6 d. A tumor-only control group received i.p. tumor cells without arginase. The survival rates of all mice were recorded. RESULTS: Survival rates were significantly lower in the i.v. group than in the i.p. group (P=0.017) or in the tumor-only control group (P=0.034). As spleen is readily exposed to i.v. arginase, its natural killer cells were studied and were found to have been significantly suppressed by arginase in vitro (P<0.005). CONCLUSION: Our results indicate that the direct inhibition of the immune system by i.v. arginase is more significant in shortening the survival of tumor-bearing mice than localized (i.p.) arginase promotion of tumor cell growth. Thus, an elevation of arginase in a patient's blood is very harmful to the host immune system, e.g. splenic natural killer cells.

The prevalence of IgE antibody reactivity against the alkaline serine protease major allergen of Penicillium chrysogenum increases with the age of asthmatic patients.

BACKGROUND: Penicillium species are prevalent airborne fungi. However, the prevalence of allergic sensitization to Penicillium antigens and the true impact of these ubiquitous fungi on atopic respiratory disorders remain to be determined. OBJECTIVE: The purpose of this study was to analyze the prevalence of immunoglobulin (Ig)E and IgG antibodies against Penicillium chrysogenum (Pen ch 13), the alkaline serine protease major allergen of P. chrysogenum, in asthmatic patients of different age groups. METHODS: Pen ch 13 was purified from a culture medium of P. chrysogenum. The reactivity of IgE and IgG antibodies to Pen ch 13 in the serum samples of 212 asthmatic patients was analyzed by immunoblotting methods. RESULTS: Sixty-nine (33%) of the 212 sera analyzed showed IgE and/or IgG immunoblot reactivity to Pen ch 13. Significant differences in the prevalence of IgE and/or IgG antibody reactivity to Pen ch 13 were found among eight different age groups of 212 asthmatic patients. The frequency of IgE-binding reactivity to Pen ch 13 increased significantly with the age of the patients. It was 7% for the group less than 10 years old and 42% for the group older than 70 years old. In addition, a significant difference between the prevalence of IgE (7%) and IgG (33%) antibodies against Pen ch 13 in the group aged 10 or less was also found. CONCLUSIONS: Our study demonstrates that IgE and IgG antibodies specific for Pen ch 13 were detected in approximately one-third of the 212 asthmatic patients analyzed. Our results suggest that allergic sensitization to Pen ch 13, and possibly to other airborne Penicillium species, is more common in older asthmatic patients.

cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum.

BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.