In the absence of a basal lamina, ameloblasts absorb enamel in a serumless and chemically defined organ culture system.

OBJECTIVES: Tooth organ development was examined in a serumless, chemically defined organ culture system to determine whether morphological and functional development was identical to that in in vivo and serum-supplemented organ cultures. METHODS: Mouse mandibular first molar tooth organs at 16 days of gestation were cultured for up to 28 days in a Tronwell culture system using a serum-supplemented or serumless, chemically defined medium. After culture, specimens were processed for assessing tooth development using ultrastructural, immunohistochemical, and mRNA expression analyses. RESULTS: In serum-supplemented conditions, inner enamel epithelial cells differentiated into secretory-stage ameloblasts, which formed enamel and reached the maturation stage after 14 and 21 days of culture, respectively. Ameloblasts deposited a basal lamina on immature enamel. Conversely, in serumless conditions, ameloblasts formed enamel on mineralized dentin after 21 days. Moreover, maturation-stage ameloblasts did not form basal lamina and directly absorbed mineralized enamel after 28 days of culture. RT-PCR analysis indicated that tooth organs, cultured in serumless conditions for 28 days, had significantly reduced expression levels of ODAM, amelotin, and laminin-322. CONCLUSIONS: These results indicate that several differences were detected compared to the development in serum-supplemented conditions, such as delayed enamel and dentin formation and the failure of maturation-stage ameloblasts to form basal laminae. Therefore, our results suggest that some factors might be required for the steady formation of mineralized dentin, enamel, and a basal lamina. Additionally, our results indicate that a basal lamina is necessary for enamel maturation.

Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

[Day surgery of early breast cancer treated with breast-conserving operation following sentinel lymph node navigation biopsy under local anesthesia].

In the early stages of breast cancer when axillary lymph node dissection is avoidable, we performed same day surgery with a breast-conserving operation following sentinel lymph node navigation biopsy under local anesthesia. Initial sentinel lymph node biopsy under local anesthesia is performed to avoid false-negative sentinel lymph node metastasis. Resected sentinel lymph nodes were examined in fixed sections by hematoxylin-eosin staining and immunohistochemistry. The enrolled subjects were 20 patients with breast cancer whose diagnoses were confirmed before treatment. Two (10.0%) patients had metastasis.

[Idiopathic mesenteric phlebosclerosis associated with long-term use of Chinese herbs: a case report].

A 59-year-old woman had been admitted to our hospital every two months for over a past year because of severe right abdominal pain. Colonoscopy revealed dark blue mucosa extending from the cecum to the transverse colon, and abdominal computed tomography showed wall thickening and linear calcification along the wall from the cecum to the transverse colon. Based on these findings, the patient was given a diagnosis of idiopathic mesenteric phlebosclerosis. Subsequently, we found that she had been a long-term user of a Chinese herbal product containing Gardeniae fructus for allergic rhinitis. After discontinuing the product, the patient has been free of abdominal pain for a year.

Intranasal administration of PACAP: uptake by brain and regional brain targeting with cyclodextrins.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent neurotrophic and neuroprotectant that is transported across the blood-brain barrier in amounts sufficient to affect brain function. However, its short half-life in blood makes it difficult to administer peripherally. Here, we determined whether the radioactively labeled 38 amino acid form of PACAP can enter the brain after intranasal (i.n.) administration. Occipital cortex and striatum were the regions with the highest uptake, peaking at levels of about 2-4% of the injected dose per gram of brain region. Inclusion of unlabeled PACAP greatly increased retention of I-PACAP by brain probably because of inhibition of the brain-to-blood efflux transporter for PACAP located at the blood-brain barrier. Sufficient amounts of PACAP could be delivered to the brain to affect function as shown by improvement of memory in aged SAMP8 mice, a model of Alzheimer's disease. We found that each of three cyclodextrins when included in the i.n. injection produced a unique distribution pattern of I-PACAP among brain regions. As examples, ß-cyclodextrin greatly increased uptake by the occipital cortex and hypothalamus, α-cyclodextrin increased uptake by the olfactory bulb and decreased uptake by the occipital cortex and striatum, and (2-hydropropyl)-ß-cyclodextrin increased uptake by the thalamus and decreased uptake by the striatum. These results show that therapeutic amounts of PACAP can be delivered to the brain by intranasal administration and that cyclodextrins may be useful in the therapeutic targeting of peptides to specific brain regions.

[Lumpectomy and sentinel lymph node navigation surgery for breast cancer under local anesthesia].

We studied and analyzed therapeutic outcomes of a radical surgery under local anesthesia for breast cancer in our department. Subjects were 42 patients with breast cancer whose diagnoses were definitely made before surgery. Indications were: localized DCIS diagnosed preoperatively; invasive carcinoma less than 3 cm in tumor diameter on ultrasound; and clinically tumors with negative axillary lymph nodes. Operative procedures included lumpectomy associated with sentinel lymph node navigation biopsy. We could perform the operation under local anesthesia in all of the 42 patients, and were not demanded to shift from local to general anesthesia. Two patients had sentinel lymph nodes metastasis. Surgical stumps were positive in 14 patients( 33.3%). None of serious complications were encountered. Today's radical operation under local anesthesia for breast cancer is a useful procedure as minimally invasive surgery as for the indications employed in this study.

IL-12 and IL-18 induction and subsequent NKT activation effects of the Japanese botanical medicine Juzentaihoto.

In this study, we first measured some cytokine concentrations in the serum of patients treated with Juzentaihoto (JTT). Of the cytokines measured interleukin (IL) -18 was the most prominently up-regulated cytokine in the serum of patients under long term JTT administration. We next evaluated the effects of JTT in mice, focusing especially on natural killer T (NKT) cell induction. Mice fed JTT were compared to control group ones. After sacrifice, the liver was fixed, embedded and stained. Transmission electron microscope (TEM) observations were performed. Although the mice receiving the herbal medicine had same appearance, their livers were infiltrated with massive mononuclear cells, some of which were aggregated to form clusters. Immunohistochemical staining revealed that there was abundant cytokine expression of IL-12 and IL-18 in the liver of JTT treated mice. To clarify what the key molecules that induce immunological restoration with JTT might be, we next examined in vitro lymphocyte cultures. Mononuclear cells isolated and prepared from healthy volunteers were cultured with and without JTT. Within 24 hours, JTT induced the IL-12 and IL-18 production and later (72 hours) induction of interferon (IFN)-gamma. Oral administration of JTT may induce the expression of IL-12 in the early stage, and IL-18 in the chronic stage, followed by NKT induction. Their activation, following immunological restoration could contribute to anti-tumor effects.

Histological and physicochemical studies of hypercalcified primear lines in the laminar bone of young calves.

There are many reports on laminar bone in various young animals during their growth periods. One of the concentric laminar bone units around the long-bone marrow periphery consists of three components: bright line, woven bone, and lamellar bone in the long-bone cortex of young calves and pigs. However, the fine structure, especially the uniform bright line or 'hypercalcified line' present in the unit-center, has not been elucidated as yet. The laminar bone of young calves was found to be initially formed from the hypercalcified lines; that is, 'hypercalcified primear' reported previously by the other authors. Such primear lines containing collagen fibrils scattered deposits of rod-like structures containing fine non-collagenous fibrils and globular structures showing no fibrils and some remnants. Osteocytes occasionally existed in the lines as well as adjacent to the lines, although no cells have been reported in the line. The cell lacunae were larger than those of woven bone and lamellar bone. The hypercalcified primear lines contained higher Ca and P content, and the molar ratio (1.78) was similar to 1.75 in the woven bone and lamellar bone. However, the physical and chemical resistances were significantly lower than that of the surrounding bone. Therefore, the hypercalcified primear lines are strongly suggested to show a lower crystallization. Further, fine structural observations of the primear-line forming cells and histochemical and immuno-histochemical investigations of the primear lines will be necessary.

Distributions of magnesium and sulphur in defect layers of incisor dentine in magnesium-deficient rats.

This study examined the mechanism governing the occurrence of defect layers of incisor dentine in Mg-deficient rats by X-ray microanalysis. Young (5 weeks of age) Wistar male rats were pair-fed semi-synthetic diets containing either control (0.05% Mg) (N = 8) or Mg-deficient (0.001% Mg) (N = 8) ingredients for 17 days. All animals were time marked with a combination of 0.1 mol nitrilotriacetato lead and 0.1 mol nitrilotriacetato zinc (2mg Pb/kg body weight) on days 0, 7 and 14 into incisor dentine. Blood samples were obtained on days 10 and 17 in order to measure Ca, Mg, P and alkaline phosphatase activity levels in serum; moreover, hypomagnesaemia and hypercalcaemia were confirmed. After the 17th day, rats were sacrificed humanely under anaesthesia and mandibular incisors were removed. Dentine formation of right mandibular incisors was assessed (time marking lines); furthermore, Ca, P, Mg and sulphur (S) concentrations as well as Ca/P molar ratio were determined in left mandibular incisors based on contiguous measurement points at 2 microm intervals from dentine pulp to dentine of the lingual aspect via X-ray analysis. Additionally, proteoglycan distribution was observed in other Mg-deficient rat dentine. These findings demonstrated decreases in body weight, incisor formation and incisor length in Mg-deficient rats. Mg and S levels increased in the defect layers, whereas proteoglycan decreased. This phenomenon was possibly attributable to condensation of Mg and S contents consequent to decreased dentine formation during Mg-deficiency and a transient increase in Mg due to transport of Mg as a result of inhibition of cell proliferation in soft tissues.

Uniformly sized molecularly imprinted polymer for atropine and its application to the determination of atropine and scopolamine in pharmaceutical preparations containing Scopolia extract.

A uniformly sized molecularly imprinted polymer (MIP) for atropine has been prepared. The MIP was prepared using 2-(trifluoromethyl) acrylic acid and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and thermal polymerization method. The selectivity factor, which is defined as the ratio of the retention factors (k) on the molecularly imprinted and non-imprinted polymers, k(imprinted)/k(non-imprinted), was 2.2 for atropine on the MIP. The obtained MIP was applied for the determination of tropane alkaloids (atropine and scopolamine) in a commercial gastrointestinal drug by a column-switching HPLC system, consisting of an MIP material as a pre-column, and a conventional cation-exchange analytical column. An interference peak was observed at the retention time of atropine derived from pre-column. However, since the peak area was less than 0.5% the peak area of atropine of a standard solution under the analytical conditions of this study (0.2 microg of atropine was loaded), this interference was negligible in the determination of atropine. On the other hand, no interference peak was observed at the retention time of scopolamine. Calibration curves of atropine and scopolamine showed good linearity in the range of 0.02-0.9 microg/ml (r=0.9999) and 0.003-0.09 microg/ml (r=0.9998), respectively. The mean recoveries of atropine and scopolamine from a placebo pharmaceutical preparation sample were 98.9 and 99.9%, respectively. The intra-day precision (measured by relative standard deviation, R.S.D. (%)) of both ingredients was less than 2.0%. The optimized column-switching system was applied successfully to the determination of atropine and scopolamine in a commercial gastrointestinal drug.