The green algal carotenoid siphonaxanthin inhibits adipogenesis in 3T3-L1 preadipocytes and the accumulation of lipids in white adipose tissue of KK-Ay mice.

BACKGROUND: Siphonaxanthin, a xanthophyll present in green algae, has been shown to possess antiangiogenic and apoptosis-inducing activities. OBJECTIVE: We evaluated the antiobesity effects of siphonaxanthin by using a 3T3-L1 cell culture system and in diabetic KK-Ay mice. METHODS: 3T3-L1 cells were differentiated with or without 5 µmol/L siphonaxanthin, and lipid accumulation and critical gene expressions for adipogenesis were examined. In vivo, 4-wk-old male KK-Ay mice were administered daily oral treatment of 1.3 mg siphonaxanthin for 6 wk and body weight, visceral fat weight, serum variables, and gene expressions involved in lipid metabolism were evaluated. RESULTS: Compared with the other carotenoids evaluated, siphonaxanthin potently inhibited adipocyte differentiation. Siphonaxanthin significantly suppressed lipid accumulation at noncytotoxic concentrations of 2.5 and 5 µmol/L by 29% and 43%, respectively. The effects of siphonaxanthin were largely limited to the early stages of adipogenesis. Siphonaxanthin significantly inhibited protein kinase B phosphorylation by 48% and 72% at 90 and 120 min, respectively. The expressions of key adipogenesis genes, including CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator activated receptor γ (Pparg), fatty acid binding protein 4 (Fabp4), and stearoyl coenzyme A desaturase 1 (Scd1), were elevated by 1.6- to 166-fold during adipogenesis. After 8 d of adipocyte differentiation, siphonaxanthin significantly lowered gene expression of Cebpa, Pparg, Fabp4, and Scd1 by 94%, 83%, 95%, and 90%, respectively. Moreover, oral administration of siphonaxanthin to KK-Ay mice significantly reduced the total weight of white adipose tissue (WAT) by 13%, especially the mesenteric WAT by 28%. Furthermore, siphonaxanthin administration reduced lipogenesis and enhanced fatty acid oxidation in adipose tissue. Siphonaxanthin was observed to highly accumulate in mesenteric WAT, and the accumulation in the mesenteric WAT was almost 2- and 3-fold that in epididymal (P = 0.14) and perirenal (P < 0.05) WAT, respectively. CONCLUSION: These results provide evidence that siphonaxanthin may effectively regulate adipogenesis in 3T3-L1 cells and diabetic KK-Ay mice.

Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells.

BACKGROUND: Bioactive marine molecules have recently received considerable attention for their nutraceutical characteristics. Considering the ever-increasing demand of nutraceuticals for anti-cancer therapy, we investigated the apoptosis-inducing effects of marine carotenoids, including siphonaxanthin, on human leukemia (HL-60) cells. METHODS: Apoptotic effects were evaluated by cell viability assay, TUNEL assay, and caspase-3 activity. The expression of apoptosis-inducing death receptor-5 (DR5), Bcl-2 and Bax were assayed by Western blot analysis, and mRNA expression of GADD45α was assayed by quantitative RT-PCR analysis. RESULTS: Siphonaxanthin potently inhibited the viability of HL-60 cells compared with the other carotenoids evaluated. In comparison with fucoxanthin, siphonaxanthin at a concentration of 20µM markedly reduced cell viability (p<0.05) as early as within 6h of treatment. The effective apoptotic activity of siphonaxanthin was observed by increases in TUNEL-positive cells, and by increased chromatin condensation in HL-60 cells. This induction of apoptosis was associated with the decreased expression of Bcl-2, and the subsequently increased activation of caspase-3. In addition, siphonaxanthin up-regulated the expression of GADD45α and DR5. CONCLUSIONS: These data suggest that the dietary carotenoid siphonaxanthin could be potentially useful as a chemo-preventive and/or chemotherapeutic agent. GENERAL SIGNIFICANCE: Our findings demonstrate for the first time the novel functional property of siphonaxanthin as a potent inducer of apoptosis in HL-60 cells.

Th17 cells contribute to nontypeable Haemophilus influenzae-specific protective immunity induced by nasal vaccination with P6 outer membrane protein and α-galactosylceramide.

Nasal vaccination is an effective therapeutic means of preventing upper respiratory infection. Recently, nasal vaccination with P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) and alpha-galactosylceramide (α-GalCer) was reported to induce NTHi-specific protective immunity. The present study investigated the role of the Th17 cells induced by nasal vaccination. Mice were immunized with P6 and α-GalCer, and their P6-specific immune responses were examined. Cytokine-producing cells were analyzed by flow cytometry, and expression of cytokines in P6-specific CD4+ T cells was determined by reverse transcription-polymerase chain reaction. Bacterial challenges were performed with live NTHi. To examine the role of Th17 cells, bacterial clearance was also evaluated after interleukin (IL)-17 neutralization. P6-specific nasal wash immunoglobulin (Ig) A and serum IgG were increased after immunization with P6 and α-GalCer. Specific IgA-producing cells increased markedly in the nasal passages (NPs) of the immunized mice. In addition to P6-specific Th1 and Th2 cells, IL-17-producing Th17 cells were induced in the NPs and spleen. Bacterial clearance was enhanced by nasal vaccination. Interestingly, impaired NTHi clearance was shown after IL-17 neutralization. These findings suggest that nasal vaccination with P6 and α-GalCer is an effective regimen for the induction of NTHi-specific protective immunity in the upper respiratory tract. In addition to antigen-specific secretory-IgA, specific Th17 cells induced by nasal vaccination contribute to protection against NTHi.

[Deep-neck infection: clinical analysis of 299 cases].

Deep-neck infection (DNI) remains potentially fatal. We retrospectively analyzed 299 surgically treated DNI cases between January 1997 and October 2007 by reviewing computed tomography (CT) results and discuss treatment and risk factors. Subjects were divided into two groups by abscess-site; peritonsillar abscess (PTA) (n=251) and deep-neck abscess (DNA) (n = 48). Age, smoking habits, body mass index (BMI) and bacteriological histories were collected from clinical records and compared by group. DNI and PTA severity parameters were C-reactive protein (CRP) titer and hospitalization length. Median subject age in DNI was 51.0 years and peak incidence in the 50s. Median subject age in PTA was 31.0 years and the peak incidence in the 20s. Smoker prevalence was higher in both groups than in normal healthy subjects. The DNI group had higher BMI and diabetes mellitus. Factors potentially most affecting illness were complications such as obesity and diabetes mellitus in DNI and age in PTA.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken.

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

Photochemical removal of NO(2) by using 172-nm Xe(2) excimer lamp in N(2) or air at atmospheric pressure.

Photochemical removal of NO(2) in N(2) or air (5-20% O(2)) mixtures was studied by using 172-nm Xe(2) excimer lamps to develop a new simple photochemical aftertreatment technique of NO(2) in air at atmospheric pressure without using any catalysts. When a high power lamp (300 mW/cm(2)) was used, the conversion of NO(2) (200-1000 ppm) to N(2) and O(2) in N(2) was >93% after 1 min irradiation, whereas that to N(2)O(5), HNO(3), N(2), and O(2) in air (10% O(2)) was 100% after 5s irradiation in a batch system. In a flow system, about 92% of NO(2) (200 ppm) in N(2) was converted to N(2) and O(2), whereas NO(2) (200-400 ppm) in air (20% O(2)) could be completely converted to N(2)O(5), HNO(3), N(2), and O(2) at a flow rate of 1l/min. It was found that NO could also be decomposed to N(2) and O(2) under 172-nm irradiation, though the removal rate is slower than that of NO(2) by a factor of 3.8. A simple model analysis assuming a consecutive reaction NO(2)-->NO-->N+O indicated that 86% of NO(2) is decomposed directly into N+O(2) and the rest is dissociated into NO+O under 172-nm irradiation. These results led us to conclude that the present technique is a new promising catalyst-free photochemical aftertreatment method of NO(2) in N(2) and air in a flow system.

[A case of scirrhous gastric cancer with peritoneal dissemination acquiring stable disease over three years by treatment with oral anticancer drug S-1].

A 71-year-old male, who complicated of abdominal distension, was diagnosed as scirrhous gastric cancer. We treated him with the oral anticancer drug S-1 and achieved long-term stable disease over three years.

Primary salivary duct carcinoma arising from the Stensen duct.

This report describes a salivary duct carcinoma (SDC) arising from the extraglandular portion of the Stensen duct. The patient was a 56-year-old man who presented with a palpable, elastic, hard mass without tenderness in the right cheek. Computed tomography revealed a tumor of the extraglandular portion of the Stensen duct. Supraomohyoid right neck dissection and total right parotidectomy were performed, and the histologic diagnosis was SDC of the Stensen duct. Postoperatively, the patient received no additional treatment. Neither recurrence nor metastasis was observed during 4 years of follow-up examination. SDC of the Stensen duct is extremely rare. To our knowledge, there is no report that describes primary SDC arising from that location. We also believe this is the first report that describes the clinical course of primary SDC arising from a Stensen duct.

Effect of low-intensity focused ultrasound on the middle ear in a mouse model of acute otitis media.

We hypothesized that low-intensity focused ultrasound (LIFU) increases vessel permeability and antibacterial drug activity in the mouse middle ear. We determined appropriate settings by applying LIFU to mouse ears with the external auditory canal filled with normal saline and performed histologic and immunohistologic examination. Acute otitis media was induced in mice with nontypable Haemophilus influenzae, and they were given ampicillin (50, 10, or 2 mg/kg) intraperitoneally once daily for 3 days with or without LIFU (1.0 W/cm(2), 20% duty cycle, 30 s). In the LIFU(+) groups receiving the 2- and 10-mg/kg doses, viable bacteria counts, number of inflammatory cells and IL-1ß and TNF-α levels in middle ear effusion were significantly lower than in the LIFU(-) groups on the same doses. Severity of AOM also tended to be reduced more in the LIFU(+) groups than in the LIFU(-) groups. LIFU application with antibiotics may be effective for middle ear infection.

Nasal immunization with plasmid DNA encoding P6 protein and immunostimulatory complexes elicits nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx.

Nasal vaccination is an effective therapeutic regimen for preventing upper respiratory infection, while DNA vaccines represent a new approach for controlling infectious diseases. Here, we examined the efficacy of nasally administered DNA vaccine on upper respiratory infections. A DNA plasmid encoding the P6 outer membrane protein of nontypeable Haemophilus influenzae (NTHi) was constructed. Mice were immunized 3 times intranasally with the DNA plasmid and Matrix-M, an immunostimulatory complex adjuvant. P6-specific immune responses were examined using purified P6 protein. Nasal-associated lymphoid tissue (NALT) CD4(+) T cells were purified and incubated with feeder cells in the presence of P6, and the expression of cytokine mRNA was examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. P6-specific nasal wash IgA and serum IgG were elevated following immunization with the DNA plasmid and Matrix-M. The number of specific IgA-producing cells increased in the nasal passages of the immunized mice. In addition to Th1 and Th2 cytokine expression, IL-17 was detected in P6-specific NALT CD4(+) T cells. Moreover, DNA vaccination enhanced bacterial clearance. These findings suggest that a successful DNA vaccination protocol has been developed for inducing in vivo immune responses against NTHi. Nasal vaccination with P6 DNA vaccine and Matrix-M might be a new effective regimen for the induction of specific protective immunity in the upper respiratory tract.

A rare case of cholesterol granuloma in the thyroid without an abnormality of lipid metabolism.

This report describes a cholesterol granuloma (CG) of the thyroid. The patient was a 79-year-old male. The mass in the thyroid was observed by chest computed tomography (CT). Initially, he had no clinical symptoms and the mass was not palpable. However, it started and became palpable and painful. He was diagnosed to have subacute thyroiditis. Although he was administered prednisolone (PSL), the mass grew larger and more solid. Then thyroid left lobectomy was performed under general anesthesia. The histological diagnosis was CG of the thyroid. After surgery his clinical course was favorable.

Nasal vaccination with P6 outer membrane protein and alpha-galactosylceramide induces nontypeable Haemophilus influenzae-specific protective immunity associated with NKT cell activation and dendritic cell expansion in nasopharynx.

The efficacy of alpha-galactosylceramide (alpha-GalCer) as a mucosal adjuvant was examined. Mice were immunized intranasally with nontypeable Haemophilus influenzae (NTHi) P6 protein and alpha-GalCer. P6-specific antibody responses in the form of P6-specific IgA in nasal washes and serum IgG titers were significantly elevated. Splenic CD4(+) T cells expressed P6-specific Th1 and Th2 cytokine mRNA. In addition, NTHi was quantified in nasal washes following NTHi challenges, and the clearance of NTHi from the nasopharynx was also enhanced. These results indicate that alpha-GalCer might be an effective mucosal adjuvant.

A single nasal dose of fms-like tyrosine kinase receptor-3 ligand, but not peritoneal application, enhances nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx.

Nasal vaccination is an effective therapeutic regimen for preventing otitis media. In the development of nasal vaccine, an appropriate adjuvant is required. In the present study, we examined the efficacy of fms-like tyrosine kinase receptor-3 ligand (Flt3L) as a mucosal adjuvant. Flt3L was administered intranasally or peritoneally to mice, which were then immunized intranasally with P6 protein of nontypeable Haemophilus influenzae (NTHi), and P6-specific immune responses were examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. Nasal application of Flt3L induced an increase in the number of dendritic cells in nasal-associated lymphoid tissue. P6-specific nasal wash immunoglobulin (Ig)A and serum IgG titers were elevated significantly after nasal immunization. Enhanced NTHi clearance from the nasopharynx was also observed. The effect of nasal vaccination with P6 combined with nasal Flt3L application was prolonged. These results indicate the potential of Flt3L as an effective mucosal adjuvant and suggest that nasal vaccination with P6 in combination with nasal Flt3L might be an effective regimen for the induction of NTHi-specific protective immunity.

Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile.

Since anti-angiogenic therapy has becoming a promising approach in the prevention of cancer and related diseases, the present study was aimed to examine the anti-angiogenic effect of siphonaxanthin from green alga (Codium fragile) in cell culture model systems and ex vivo approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring, respectively. Siphonaxanthin significantly suppressed HUVEC proliferation (p<0.05) at the concentration of 2.5 µM (50% as compared with control) and above, while the effect on chemotaxis was not significant. Siphonaxanthin exhibited strong inhibitory effect on HUVEC tube formation. It suppressed the formation of tube length by 44% at the concentration of 10 µM, while no tube formation was observed at 25 µM, suggesting that it could be due to the suppression of angiogenic mediators. The ex vivo angiogenesis assay exhibited reduced microvessel outgrowth in a dose dependent manner and the reduction was significant at more than 2.5 µM. Our results imply a new insight on the novel function of siphonaxanthin in preventing angiogenesis related diseases.

Tonsillar focal infectious disease involving IgA nephropathy, pustulosis, and ossification.

We report a rare case of IgA nephropathy (IgAN), that was considered as showing tonsillar focal infection, involving pulmoplantar pustulosis (PPP), and sternocostoclavicular hyperosteosis (SCCH). A 53-year-old man with a 3-year history of PPP had hematuria and proteinuria, and he sometimes had anterior chest pain. He was also diagnosed with IgAN and SCCH. We performed tonsillectomy as a treatment. The tonsillectomy was done with the patient under general anesthesia, and this treatment was followed by steroid therapy. Interestingly, all the symptoms of IgAN, PPP, and SCCH were alleviated 6 months after the tonsillectomy. Thus, tonsillectomy and steroid therapy may be effective and could be considered as treatment for these diseases.