Fumiyuki Kuwata

Characterization of a Monoclonal Antibody Recognizing Small Collagenous Proteins in Fetal Bone

A monoclonal antibody (MBP-322) that recognizes two small collagenous, apatite-binding (SCAB) proteins associated with the mineral phase of fetal bone, has been prepared. The SCAB proteins, which are quantitatively extracted from bone with EDTA, have been shown by immunotransfer analyses to have MrS of 28K and 25K and both were selectively degraded by bacterial collagenase. Amino acid analysis of the collagenase-digested protein revealed a hypro:pro:gly ratio of approximately 0.5:1:0.7 for both proteins and indicated that one-third of the protein could have a collagen-like sequence. The SCAB proteins, unlike other collagens and collagen fragments tested bound quantitatively to hydroxylapatite in the presence of 4M guanidine hydrochloride and appear to be unique to bone. The antibody, however, was not specific for the SCAB proteins and showed comparable immunoreactivity against denatured alpha 1 chains of types I, II and III collagens and the alpha 2 chains of types I and V collagens but not type IV collagen nor native collagens I-V. The epitope was further localized to the CB6 fragment in the alpha 1(I) chain and the CB5 fragment of alpha 1(III) chain, and was present in both the TCA and TCB fragments of alpha 2(I). Despite the immunological reactivity, the properties of the SCAB proteins were not consistent with their being derived from known collagen types. Immunocytochemical staining of permeabilized bone cells with MPB-322 showed a perinuclear, punctate staining pattern in most cells with some cells showing specific nuclear staining. In non-permeabilized cells, the antibody stained various sized spherical particles, many of which were closely associated with the cell surface. Immunoblots of cell proteins revealed a number of immunoreactive proteins sensitive to collagenase digestion including two proteins with MrS similar to the SCAB proteins. The MBP-322 antibody appears useful for identifying sequence homology in various collagens, and for recognizing denatured collagen and specific collagen fragments in tissues, as well as being important for the further characterization of the SCAB proteins.

Enzyme Patterns of Glycolysis in Rabbit Gingiva and Effects of pH on the Patterns

The specific activities of individual enzymes associated with glycolysis in rabbit gingiva were systematically evaluated, and the enzyme patterns at pH 7.4 were established on the glycolytic and gluconeogenic pathways. As a result, it was suggested that there was no capability for gluconeogenesis. The effects of alkaline pH values on the enzyme patterns appeared to lead to the overall stimulation of glycolysis.

Assessment of quality and interpretation of panoramic radiographs obtained in the Lao People’s Democratic Republic as part of a teleradiology collaboration with Japan

As part of quality assessment of a teleradiology program we evaluated the validity of patient information received, the quality of panoramic radiography imaging in Laos, and the ability of a Laotian radiologist to detect temporomandibular joint abnormalities. The amount of patient information gathered from 2,021 scans of panoramic radiographs was evaluated by triage before image diagnosis. Among the radiographs from 2,021 patients, primary triage indicated that there was insufficient information for 794 (39.3%) patients. Secondary triage to assess imaging failure included 1,227 radiographs, four of which were excluded from imaging diagnosis because of unacceptable image flaws. In total, 2,446 joints from 1,223 radiographs were evaluated for temporomandibular joint abnormalities in order to compare the image interpretation abilities of Laotian and Japanese radiologists. The kappa coefficient was 0.836 (P < 0.01) for the agreement between the two observers in detecting temporomandibular joint abnormalities on radiographs. We conclude that additional efforts are needed in order to overcome the challenges of maintaining quality in imaging techniques and diagnoses in Laos.

Properties of malate dehydrogenase isozymes in rabbit dental pulp.

Delbruck et al. (Biochem Z 331:273, 1959) first demonstrated a multiplicity of malate dehydrogenases (MDH:EC 1.1.1.37) in tissues. Thorne et al. (Biochim Biophys Acta 42:175, 1962) isolated and characterized mitochondria and supernatant MDH (M-MDH and S-MDH) from pig or beef heart. However, the isozymes of MDH have not been reported in dental pulp. In this annotation, it is shown that MDH in rabbit dental pulp can be separated into three anionic isozymes and one cationic isozyme by electrophoresis. The enzymatic properties of each isozyme are described. Dental pulp (wet weight 500 mg) from molars of albino rabbits (weight approx. 2 kg) was homogenized in 5 ml of 0.25 M sucrose with a Teflon homogenizer. The mitochondria and supernatant fractions were obtained by the method of Hogeboom et al. (J Biol Chem 172:619, 1948). Each fraction was applied on starch-gel § (1 x 1 x 2 5 cm) previously equilibrated with 0.1 M veronal buffer, pH 8.6. After 16 hours of electrophoresis (conditions: 8 V/cm, 4 mA at 40C), the gel was cut into blocks (1 x 1 x 0.5 cm). Then each block was extracted with 5 in! of 0.15 M KCl during two hours of occasional stirring. The incubation assay mixture for MDH contained 20 pmole phosphate buffer, pH 7.6; 0.4 ,umole oxalacetate; and 0.08,mole sodium NADH, in 0.4 ml. The reaction was carried out at 370C for 30 min and terminated by the addition of 0.4 ml of 10% perchloric acid. After 15 min from the termination, 2.0 ml of 10 N NaOH was added to be reincubated for one h at 370C. The fluorescence originating from the NAD formed was determined by a fluorescence spectrophotometert

Predicting oral malodour based on the microbiota in saliva samples using a deep learning approach

BackgroundOral malodour is mainly caused by volatile sulphur compounds produced by bacteria and bacterial interactions. It is difficult to predict the presence or absence of oral malodour based on the abundances of specific species and their combinations. This paper presents an effective way of deep learning approach to predicting the oral malodour from salivary microbiota.MethodsThe 16S rRNA genes from saliva samples of 90 subjects (45 had no or weak oral malodour, and 45 had marked oral malodour) were amplified, and gene sequence analysis was carried out. Deep learning classified oral malodour and healthy breath based on the resultant abundances of operational taxonomic units (OTUs)ResultsA discrimination classifier model was constructed by profiling OTUs and calculating their relative abundance in saliva samples from 90 subjects. Our deep learning model achieved a predictive accuracy of 97%, compared to the 79% obtained with a support vector machine.ConclusionThis approach is expected to be useful in screening the saliva for prediction of oral malodour before visits to specialist clinics.

An Enzymological Profile of the Production of Lactic Acid in Caries-Associated Plaque and in Plaque Formed on Sound Surfaces of Deciduous Teeth

Specific activities of lactate dehydrogenase (LDH) and its fructose 1,6-bisphosphate (FBP) dependency in the small amount of plaque formed on deciduous teeth were determined and compared between those from carious lesions and from sound surfaces. Although the caries-associated plaque showed a higher production of lactic acid than the plaque formed on sound surfaces, it occurred only when sufficient FBP was present. The proportion of activity of FBP-dependent LDH relative to that of total LDH varied much more in the caries-associated plaque than in the plaque formed on sound surfaces. Hence, the varying activity of FBP-dependent LDH might be a distinguishing feature of caries-associated plaque compared with plaque formed on sound surfaces.