[Validation Study of Analytical Method for Determination of Amnesic Shellfish Poison in Bivalves].

Amnesic shellfish poison (ASP) is regarded as one of the shellfish poison groups in the EU, though it is not subject to regulation in Japan. We have developed an analytical method of ASP based on the report by Hatfield et al. and other methods. Validation studies were carried out with certified compositional reference materials (CRM). Performance parameters were estimated based on 17 analytical results. The estimate of trueness was 97.5%, and the estimate of intralaboratory reproducibility (RSD) was 1.5%. The HorRat(r) value was 0.16. These performance parameters meet the criteria in the Codex Procedural Manual. Furthermore, internal quality control was performed by using the CRM. The action limits were set based on the performance parameters of the method. Most of the results of the internal quality control were within the action limit range. The results confirmed that the quality of the analyses was well maintained. The purpose of the analytical method is to confirm that the level of ASP in scallop is less than 4.6 mg/kg. The applicability of the analytical method to scallops was confirmed by using spiked samples.

Intra-surgical vs. radiographic bone level assessments in measuring peri-implant bone loss.

OBJECTIVE: To evaluate the accuracy between the intra-surgical and the peri-apical radiographic measurements of bone loss at implant with peri-implantitis. MATERIALS AND METHODS: A total of 46 Brånemark implants in 24 patients with diagnosis of peri-implantitis were included in the study. The amount of peri-implant bone loss occurred at those implants was measured during peri-implant surgery and compared to the radiographic bone loss measured by three independent examiners. RESULTS: The mean bone loss measured on radiographs underestimated the intra-surgical bone loss at the correspondent sites (0.7 mm at the mesial and 0.6 mm at the distal sites); this underestimation was found to be a consistent finding in all the three examiners. Only 21% of the radiographic measurements corresponded to the clinical bone loss assessments, while an over- and underestimation within a range of ± 1-2 mm was recorded in 57% of the cases. There was a moderate positive linear correlation between the radiographic measurements and the clinical bone loss for mesial and distal sites (r = range 0.58-0.65). The variability between the three examiners in the radiographic measurements was frequently on the range of ± 1-2 mm. CONCLUSION: The radiographic measurements of bone loss at implant affected by peri-implantitis often underestimated the clinical bone loss occurred at the implants. A difference of about ± 1-2 mm in the estimation of radiographic bone loss could be merely assigned as inter-examiner different assessments.

Efficacy and safety of a therapeutic apparatus using hydrogen peroxide photolysis to treat dental and periodontal infectious diseases.

The present study aimed to evaluate the acute locally injurious property of our most current hydroxyl radical generation system by hydrogen peroxide (H2O2) photolysis. This system, which releases 3% H2O2 with a 405-nm laser, was developed in our laboratory for the treatment of dental and periodontal infectious diseases. First, the hydroxyl radical yield generated by H2O2 photolysis was examined by applying an electron spin resonance-spin trapping technique. Second, the bactericidal effect of the device was examined under a simulant condition in which Streptococcus mutans, a pathogenic bacterial species that causes caries, was irrigated with running 3% H2O2 concomitantly with laser irradiation. Finally, the acute topical effect of the model apparatus on rat palatal mucosa was evaluated by histological examination. We found that the hydroxyl radical yield was dependent upon laser output power. The bacterial count was substantially reduced within as little as 3 min. No abnormal findings were observed in the palatal mucosa, even when rats received three treatments of 3% H2O2 with laser irradiation at an output power of 40 mW. These results suggest that our apparatus has the ability to kill bacteria via hydroxyl radical generation and is safe to use at the lesion site of dental and periodontal infectious diseases.

[Electrophoretic Analysis of Abnormal Data on Serum Protein Electrophoresis].

We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the significance of the complexes was not clear. We describe a non-diabetic patient with IgA type M-protein whose serum fructosamine and glycoalbumin levels were elevated. On electrophoresis of the serum protein of the patient, the albumin band shifted to the cathode side. The abnormal precipitin arc of IgA-albumin complexes was detected by immunoelectrophoresis. To elucidate the mechanism of IgA-albumin complexes, we analyzed their properties using immunoelectrophoresis, Western blotting, and two-dimensional gel electrophoresis. The macromolecularized albumin spots were demonstrated by two-dimensional Western blotting with antiserum to human albumin of the patient's serum. Moreover, the IgA-albumin complexes were dissociated on treatment with 2-mercaptoethanol. It can be considered that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the albumin binding site is located near the hinge region (311Cys) of the IgA molecule and involves the free SH group, thought to be present in the α-chain.

An immunoglobulin A1 that inhibits lactate dehydrogenase activity, with reversal of inhibition by addition of NADH.

We discovered a patient with low serum lactate dehydrogenase (LD) activity and an abnormal LD isozyme pattern. We analyzed the patient's LD inhibitor using electrophoresis, affinity chromatography, and immunochemical technologies. The LD activity of the patient's serum was inhibited more strongly at 4 degrees C than at 37 degrees C. The decrease of LD activity was more marked in a mixture of the patient's serum with purified LD5 than in that with purified LD1. The immunoglobulin responsible for LD inhibition was an IgA1-lambda. The LD inhibition by the patient's IgA1 was blocked by reduction and alkylation and by NADH. Polymerization of the patient's IgA1 might play an important role in its interaction with LD. Moreover, the possibility exists that part of the patient's IgA1 molecule fits into a pocket of LD in instead of NADH. This is the first report of NADH reversing such LD inhibition.

[Characterization of anti-DNA antibody on IgG3-kappa type M-protein in a patient with rheumatoid arthritis].

A 66 year-old male patient with rheumatoid arthritis, was admitted due to bronchial pneumonia. Anti-nuclear antibody and anti-DNA antibody were both detected in the patient's serum and many LE-like cells were found in the pleural effusion. In addition, M-proteins(IgG-kappa type, IgM-kappa type) were detected in both serum and pleural effusion by immunofixation electrophoresis. Purified IgG fraction by gel filtration(Superdex 200HR) showed high titer of anti-nuclear antibody(x160) and anti-DNA(39.2 AU/ml) antibody. Furthermore, anti-DNA antibody was detected in the fraction III(fraction of M-protein) by ion-exchange chromatography. In conclusion, these results indicate that M-protein(IgG3-kappa type) contains anti-DNA antibody and it is suggests that M-protein induces the production of LE like cell in this case.

Complexes of gelatinases and tissue inhibitor of metalloproteinases in human seminal plasma.

Previously reported data have indicated the existence of two kinds of matrix metalloproteinases (MMP-2 and MMP-9) in human seminal plasma (Shimokawa et al, 2002). Here we report the existence of complexes of gelatinases and tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in human seminal plasma. After the seminal plasma supernatant was separated on a gel-filtration column chromatography of GCL-2000-sf-cellulofine. Western blot analysis showed these proteins were recognized by two antibodies to TIMP-1 and TIMP-2, but not to TIMP-3 or TIMP-4. These bands were consistent with standard recombinant full-length TIMP-1 and TIMP-2 proteins. These bands had molecular weights of approximately 29 and 21 kd for TIMP-1 and TIMP-2, respectively. These proteins existed as complexes of proMMP-9/TIMP-1, proMMP-2/TIMP-2, MMP-2/TIMP-2, free TIMP-1, and TIMP-2 in human seminal plasma. The partially free TIMPs were degradated by some proteinases in human seminal plasma. These results indicate two kinds of TIMPs (TIMP-1 and TIMP-2) and their complexes with progelatinases in human seminal plasma.

Identification of complexes of gelatinase A and tissue inhibitor of metalloproteinase-2 in human follicular fluid.

Background and Aims: Ovulation involves considerable tissue remodeling in normal ovarian function. These processes are expected to involve matrix metalloproteinases (MMP). Follicular rupture is caused by the degradation of the basement membrane between the thecal and granulose layers, as well as disruption of the extracellular matrix (ECM) at the site of rupture. We report on the existence of the complexes of progelatinase A (proMMP-2), MMP-2 and a tissue inhibitor of metalloproteinase-2 (TIMP-2) using zymographic and immunological techniques in human follicular fluid (HFF). Methods and Results: Partial purification of the complexes was achieved by using gelatin affinity column chromatography. The peak (tubes 68-73) in this chromatography showed gelatinase activities by gelatin-zymography, and also an inhibition by EDTA (metalloproteinase inhibitor). The molecular weights of the gelatinase activities were approximately 72 and 67 kDa, and were consistent with standard proMMP-2 and MMP-2, as found by using gelatin-zymography. Similarly, the band in this peak was consistent with standard recombinant full-length TIMP-2, as found by the use of western blot analysis, and the molecular weight of the band was approximately 21 kDa. Conclusion: As proMMP-2, MMP-2 and TIMP-2 exist in the peak from the gelatin affinity column, we expected that these form the complexes. These results indicate that the complexes of proMMP-2/TIMP-2 and MMP-2/TIMP-2 exist in HFF. (Reprod Med Biol 2003; 2: 115-119).

Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma.

We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS-PAGE, gelatin-zymography and Western blot to be approximately 92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.