Isolation and characterization of microsatellites in Brassica rapa L.

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.

Enhancement of bone regeneration using resorbable ceramics and a polymer-ceramic composite material.

The aim of this experimental study was to evaluate the use of resorbable implants for the repair of nonloaded skeletal defects. Porous ceramic implants of alpha-TCP, of glass-ceramic, and of solid composite implants of glass-ceramic/polylactic acid 8 mm in diameter and 2 mm in thickness were fabricated and implanted pressfit into biparietal, full-thickness defects of the calvaria of 60 adult rats. Twenty rats received unfilled defects and served as controls. Fluorochrome labeling of bone formation was performed during the observation period. Five animals from each group were evaluated after 6, 13, 26, and 52 weeks. The control defects showed incomplete regeneration, with bone formation extending 1.66 mm, on average, into the defect after 52 weeks. In the group of alpha-TCP implants, histologic evaluation indicated that the bone formed during initial stages had undergone resorption later on, so that bone repair after 52 weeks was not significantly enhanced, with an average depth of 1.83 mm of bone ingrowth. The glass-ceramic implants exhibited extensive bone formation and nearly complete repair of the calvarial defect, with 3.90 mm of bone ingrowth into the implant pores. Degradation of the ceramic was nearly complete, with a few remaining particles surrounded by soft tissue. The composite implants showed a negligible bone ingrowth of 0.63 mm, on average. Soft tissue had invaded the polylactic acid implant body, but no bone formation had taken place at the surface of the embedded ceramic particles. Degradation of the polymer was not complete after 52 weeks. It is concluded that the balance between degradation and bone formation is delicate and that chemical events and cellular reaction during degradation may counteract complementary bone ingrowth.

Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose.

The crystals of beta-amylase from Bacillus cereus belong to space group P21 with the following cell dimensions: a = 57.70 A, b = 92.87 A, c = 65.93 A, and beta =101.95 degrees. The structures of free and maltose-bound beta-amylases were determined by X-ray crystallography at 2.1 and 2.5 A with R-factors of 0.170 and 0.164, respectively. The final model of the maltose-bound form comprises 516 amino acid residues, four maltose molecules, 275 water molecules, one Ca2+, one acetate, and one sulfate ion. The enzyme consists of a core (beta/alpha)8-barrel domain (residues 5-434) and a C-terminal starch-binding domain (residues 435-613). Besides the active site in the core where two maltose molecules are bound in tandem, two novel maltose-binding sites were found in the core L4 region and in the C-terminal domain. The structure of the core domain is similar to that of soybean beta-amylase except for the L4 maltose-binding site, whereas the C-terminal domain has the same secondary structure as domain E of cyclodextrin glucosyltransferase. These two maltose-binding sites are 32-36 A apart from the active site. These results indicate that the ability of B. cereus beta-amylase to digest raw starch can be attributed to the additional two maltose-binding sites.

Reconstruction of calvarial defects by bioresorbable ceramics: an experimental study in rats.

This study evaluated bioresorbable ceramics in the reconstruction of calvarial defects. Full-thickness defects were made in the calvaria of 40 adult Sprague-Dawley rats (350-450 g) with a standard 8-mm trephine drill. Three different materials were used for defect repair: (a) pure alpha-tricalcium phosphate (TCP), (b) surface-treated glass ceramic, (c) surface-treated glass ceramic plus 70/30 L/DL polylactic acid (volume ratio 45/55). The implants were pellets of 7.9 mm diameter and 2 mm thickness and were inserted press fit into the calvarial defects. Each of these materials was inserted into ten animals. Five animals were evaluated each after 6 weeks and 26 weeks. For each interval there was a control group of five animals. After 6 weeks the control defects exhibited negligible bone regeneration at the defect margins but showed substantially more bone regeneration after 26 weeks extending up to 2.5 mm into the defect space. The TCP specimens showed a number of multinuclear cells on the material surface and direct bone/implant contact in a few locations but no signs of gross degradation or volume reduction after 6 weeks. The amount of bone ingrowth and cellular behavior had not changed after 26 weeks with resorption still going on. Glass ceramic implants by contrast appeared to be even better tolerated after 6 weeks with remarkable bone ingrowth and broad osseous fixation of the pellet to the defect margins and beyond, while highly vascular connective tissue filled the remaining pores of the implant. After 26 weeks the material had been extensively degraded, leaving behind only a few remnants, which were surrounded by seams of highly vascularized and cell-rich resorptive connective tissue with newly formed bone tissue nearly bridging the defect. The polylactic acid/ceramic composite implants showed hardly any tissue ingrowth or degradation either after 6 or after 26 weeks. Hence all tested materials appeared to be well tolerated at the site of implantation. However, gradual replacement by bone ingrowth tended to occur only in implants without polylactic acid.

Hypertriglyceridemia associated with tumor necrosis factor-alpha in hamster cheek-pouch carcinogenesis.

We demonstrated for the first time that 9,10-dimethyl-1,2-benzanthracene (DMBA)-treated hamsters showed hypertriglyceridemia followed by cachexia. Hypertriglyceridemia is believed to be caused in part by the decreased lipoprotein lipase (LPL) activity, and by cytokines such as tumor necrosis factor (TNF)-alpha. In addition, TNF-alpha action is associated with the LPL activity. Therefore, we determined the content of triglyceride (TG), LPL, and TNF-alpha in the serum from DMBA-treated hamsters. Elevated TG concentration in the serum of tumor-bearing hamsters was more remarkable and preceded the increase in other lipids, whereas the activity of LPL, the key enzyme of TG metabolism in vivo, was drastically reduced. TNF-alpha, known as an endogenous inhibitor of LPL activity, was detected in both the sera and the extract of tumors from DMBA-treated hamsters, whereas it was not detectable in any control samples. Pre-incubation of control sera with exogenous recombinant human TNF-alpha resulted in a potent inhibition of endogenous LPL activity in a dose-dependent manner in vitro. Therefore, the presence of TNF-alpha might lead to the increase in plasma TG mediated by LPL in tumor-bearing hamsters.

Salivary beta 2-microglobulin in NZB/WF1 mice.

Unstimulated parotid and submandibular/sublingual saliva from New Zealand black/WF1 (NZB/WF1) mice as collected by microcapillary (1 microliter), and the content of beta 2-microglobulin (beta 2-MG) determined by a sandwich enzyme immunoassay. Salivary beta 2-MG contents of control C57BL/6 and NZB/WF1 mice (45 weeks of age) were 0.68 +/- 0.18 micrograms/ml and 1.42 +/- 0.21 micrograms/ml (mean +/- SD), respectively, from the parotid gland and 0.62 +/- 0.17 micrograms/ml and 1.34 +/- 0.21 microliters/ml, respectively, from the submandibular sublingual glands. However, the concentration of beta 2-MG was not increased in the NZB/WF1 mice at 5 and 20 weeks of age. Saliva from NZB/WF1 mice (45 weeks old) was fractionated by micro two-dimensional gel electrophoresis; it exhibited both qualitative and quantitative changes in protein composition in comparison to the two-dimensional at 5 weeks of age. These observations parallel those in saliva from patients with Sjögren's syndrome.

Increased beta 2-microglobulin in both parotid and submandibular/sublingual saliva from patients with Sjögren's syndrome.

Samples of unstimulated saliva from patients with sialoadenopathy were collected by microcapillary tube (1 microliter), and their beta 2-microglobulin (B2-MG) content determined by a sandwich enzyme immunoassay. A higher than normal (control) concentration of the globulin was present in both parotid and submandibular/sublingual saliva from the patients with Sjögren's syndrome but not in the samples from the patients with sialoadenitis or diabetes mellitus. The increase in B2-MG in saliva from patients with Sjögren's syndrome may reflect that immunolopathological events are important in the degeneration of both glands in this disease. Therefore, the determination of B2-MG in saliva may be a simple, non-invasive technique for confirming the diagnosis of Sjögren's syndrome as an autoimmune disease.

Two-dimensional electrophoresis of human salivary proteins from patients with sialoadenopathy.

Unstimulated saliva was fractionated by micro two-dimensional gel electrophoresis, and the proteins visualized by silver staining and immunostaining. The subjects with Sjögren's syndrome exhibited both quantitative and qualitative alterations in the protein composition of the saliva not only from the parotid gland but also from the submandibular/sublingual glands.

Hyperkeratosis and atypical proliferation appearing in the lining epithelium of a radicular cyst. Report of a case.

It is widely accepted that radicular cysts (apical periodontal cysts) are commonly lined with stratified squamous epithelium without keratin formation. However, we identified a case of maxillary radicular cyst with remarkable keratinization and atypical proliferation of the lining epithelium among the 207 radicular cyst cases seen at our department. Histopathological and clinical findings of these cysts were reviewed.

Enhancement by cyclosporin A of metastasis from hamster cheek pouch carcinoma.

Daily intravenous administration of 50 mg/kg cyclosporin A (CsA), after excision of cheek pouch tumors induced with 9,10-dimethyl-1,2-benzanthracene (DMBA), enhanced the incidence of cervical lymph node metastasis to 93%, a significant increase over the rates observed for animals receiving a dosage of 25 mg/kg (43%), and for control animals receiving no CsA (40%). No significant effect was evident on the growth rates of the metastasized tumour cells in the lymph node.

Lung and cervical lymph-node metastasis after cortisone enhancement in hamster cheek-pouch carcinogenesis.

The administration of hydrocortisone after excision of DMBA-induced tumours in hamster cheek pouch produced cervical lymph-node metastases in 9 out of 13 animals (69 per cent), and resulted in lung metastasis in 1 animal. In the control group, which received no cortisone but had tumours cut from the pouch, 4 out of 9 animals had cervical lymph-node metastases (44 per cent).

Regional lymph node metastasis created by partial excision of carcinomas induced in hamster cheek pouch with 9,10-dimethyl-1,2-benzanthracene.

The feasibility of using the hamster cheek pouch/dimethyl-benzanthracene (DMBA) system as an experimental model of lymphatic metastasis was investigated. Forty male Syrian golden hamsters treated with DMBA were divided into two equal groups--one with surgical excision of their tumors and a control group without tumor excision. In the excision group, the animals received three applications/week to the left cheek pouch of 0.3% DMBA in acetone for 14 weeks. Following a three-week observation period, the tumors in the pouch were excised at their base, and the animals were killed after four weeks of further observation. In the control group, the animals were treated for 14 weeks in a manner similar to that used for the excision group, left for seven weeks without treatment, and then killed. Cheek pouches with tumors and cervical lymph nodes were processed for histological examination. All of the animals, both with and without metastasis, had borne squamous cell carcinomas (SCC) in their treated cheek pouches. Histologically, seven out of 16 animals in the excision group showed metastatic deposits of SCC confined to the left cervical lymph nodes, while in the control group, metastasis was not found in any of the 19 animals with SCC in their cheek pouches. The results demonstrate that surgical excision of the hamster cheek pouch carcinoma is efficient in producing unequivocal lymph node metastasis.

Analysis and identification of human parotid salivary proteins by micro two-dimensional electrophoresis and Western-blot techniques.

Using this method, without denaturing agents, over 60 protein components in 2 microliter (about 4 micrograms of protein) of unconcentrated parotid saliva could be detected. After two-dimensional separation, the spots of albumin, secretory IgA, IgG, acid phosphatase, amylase and plasma-originating protein were identified by antibodies with the Western-blot technique.

Two-dimensional electrophoresis in the analysis of a mixture of human sublingual and submandibular salivary proteins.

Complex protein mixtures of unstimulated human sublingual and submandibular saliva were fractionated by two-dimensional (2-D) gel electrophoresis, visualized by silver staining and then analysed by immunostaining. Specific proteins were identified by incubation with specific antibody and peroxidase-conjugated second antibody (Western blot). Electrophoresis and silver staining revealed over 50 protein components in 2 microliter of unconcentrated mixture. The Western-blot technique allowed detection of protein spots of plasma origin when an antibody against whole serum was used, but only the albumin spot could be found. Albumin, secretory IgA, acid phosphatase and alpha-amylase were identified with specific antibodies.