Involvement of a lysine-specific cysteine proteinase in hemoglobin adsorption and heme accumulation by Porphyromonas gingivalis.

The oral anaerobic bacterium Porphyromonas gingivalis, a major pathogen of advanced adult periodontitis, produces a novel class of cysteine proteinases in both cell-associated and secretory forms. A lysine-specific cysteine proteinase (Lys-gingipain, KGP), as well as an arginine-specific cysteine proteinase (Arg-gingipain), is a major trypsin-like proteinase of the organism. Recent studies indicate that the secreted KGP is implicated in the destruction of periodontal tissue and the disruption of host defense mechanisms.

A longitudinal observation of the postnatal craniofacial growth in artificial monozygotic twin mice.

An artificial monozygotic twin mouse produced by bisecting a 16-cell stage embryo with a fine glass-needle and then transferring a pair of demi-embryos together into the right uterus of a pregnant recipient female mouse after a 24 hr in-vitro cultivation allowed us to examine the postnatal growth changes in monozygotic twin mice. With regard to the preimplantational growth process of the embryo, we investigated the success rate of bisecting an embryo into paired demi-embryos, the developmental ability of the bisected demi-embryo up to the early blastocyst, and the production rate of such monozygotic twin mice. The items examined in the postnatal growth process of the monozygotic twin mouse were the growth curve of the twin weight and the craniofacial size of the monozygotic twin mouse by cephalometric observations at the 3rd, 10th, 21st, 42nd, 70th, and 100th postnatal days, respectively.

Biological functions of UDP-glucose synthesis in Streptococcus mutans.

A gene encoding glucose-1-phosphate uridylyltransferase (EC 2.7.7.

Characteristics of protrusive and lateral excursions of the mandible in children with the primary dentition.

This study characterizes the mandibular protrusive and lateral excursions of children with primary dentition. With use of a Selspot system, the protrusive and lateral excursions of nine children with the primary dentition and nine adults with the permanent dentition were measured and compared. This system was able to analyse the simultaneous movements of multiple points on the mandible of a subject in three dimensions.

Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.

Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme.

Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease.

The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed "Lys-gingipain," was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography.

Influence of the discontinuation of a school-based, supervised fluoride mouthrinsing programme on the prevalence of dental caries.

Objective: Since 1984 a weekly supervised fluoride mouthrinsing programme has been implemented for schoolchildren in Hisayama, Japan. Consequently, four years after the start of the programme the DMFT index for 12.5-year-old schoolchildren in Hisayama decreased to a value of less than 3.

Binding of salivary glycoprotein-secretory immunoglobulin A complex to the surface protein antigen of Streptococcus mutans.

The interaction between a surface protein antigen (PAc) of Streptococcus mutans and human salivary agglutinin was analyzed with a surface plasmon resonance biosensor. The major component sugars of the salivary agglutinin were galactose, fucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Binding of salivary agglutinin to PAc was calcium dependent and heat labile and required a pH greater than 5.

Identification of a genetic locus essential for serotype b-specific antigen synthesis in Actinobacillus actinomycetemcomitans.

A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized. Western blot analysis showed that Escherichia coli DH5alpha, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A. actinomycetemcomitans Y4.

A longitudinal study of the postnatal maternal effect on the craniofacial growth of mouse offspring by cross-nursing.

A cross-nursing experiment was conducted to examine the nursing dam strain effect on the postnatal growth of a newborn mouse offspring by simultaneously using larger DDD strain mice and smaller C57BL strain ones. A periodical cephalometric observation of the postnatal craniofacial growth of the offspring was longitudinally made from birth up to the 100th day of life in addition to measuring the offspring weight. According to multivariate statistical analyses, the following results were obtained: 1) The mean body weight of the DDD offspring cross-nursed by the C57BL dam was significantly lighter than that of the DDD offspring self-nursed by the DDD dam throughout the whole experimental period except at birth and on the 100th day.

Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans.

We had isolated three genes (rmlA, rmlB, and rmlC) involved in dTDP-rhamnose synthesis in Streptococcus mutans and found that three genes were insufficient for dTDP-rhamnose synthesis (Y. Tsukioka, Y. Yamashita, T.

Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans.

To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc.

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria.

Identification of a frameshift mutation resulting in premature termination and loss of cell wall anchoring of the PAc antigen of Streptococcus mutans GS-5.

Most strains of Streptococcus mutans possess a 190-kDa protein antigen (PAc) on their cell surfaces, while strain GS-5 produces extracellularly a 155-kDa PAc protein. The pac gene of strain GS-5 consists of 3,477 bp and codes for a protein of 1,158 amino acids. One insertion of an adenine into the 3,469th, 3,470th, or 3,471st position from the start codon results in a frameshift mutation at codon 1157 with subsequent termination after 3 additional codons.

Increased expression of cathepsins E and D in neurons of the aged rat brain and their colocalization with lipofuscin and carboxy-terminal fragments of Alzheimer amyloid precursor protein.

Age-related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Nonlysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age.

The gnd gene encoding a novel 6-phosphogluconate dehydrogenase and its adjacent region of Actinobacillus actinomycetemcomitans chromosomal DNA.

A 10-kb DNA fragment containing the gnd gene from Actinobacillus actinomy-cetemcomitans Y4 was isolated and sequenced. The structural gnd gene codes for 6-phosphogluconate dehydrogenase that consists of 484 amino acids. In contrast to the gnd gene in Escherichia coli, Salmonella typhimurium, or Klebsiella pneumoniae, the gnd gene of A.

Isolation and characterization of the dnaKJ operon from Actinobacillus actinomycetemcomitans.

The dnaKJ operon of Actinobacillus actinomycetemcomitans Y4 was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two conserved regions in DnaK/hsp70 proteins and sequenced by inverse PCR. The sequenced region was shown to contain two open reading frames coding for proteins analogous to DnaK and DnaJ.

A clinical study of the relationship between occlusal curvature and craniomandibular disorders.

The occlusal curvature should be harmonized with stomatognathic function, but excessive occlusal curvatures are found in some craniomandibular disorder patients. Forty healthy subjects and 95 patients with craniomandibular disorder (50 with clicking, 25 with locking, and 20 with myofascial pain dysfunction syndrome) were evaluated to investigate the functional significance of the occlusal curvature. Anteroposterior and lateral occlusal curvature were measured using the least-square approximation of the mandibular buccal cusps in a second-order quadratic and modification of Monson's 4-inch sphere.

Molecular and genetic analysis of multiple changes in the levels of production of virulence factors in a subcultured variant of Streptococcus mutans.

We previously isolated a variant strain, Xc100L, which shows decreased production of a surface protein antigen with a molecular mass of 190 kDa (PAc), after repeated subculturing of Streptococcus mutans strain Xc [Koga, T. et al. (1989) J.

Cloning and sequencing of the gene encoding a novel lysine-specific cysteine proteinase (Lys-gingipain) in Porphyromonas gingivalis: structural relationship with the arginine-specific cysteine proteinase (Arg-gingipain).

Lys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5,169-bp open reading frame encoding 1,723 amino acids with a calculated molecular mass of 218 kDa.

A genetic study of dentin growth in the mandibular second and third molars of male mice.

Although the influence of genetic and environmental factors on the completed crown size of mandibular molars in mice is well known, such influence on the growth process of the mandibular second and third molars of mice has not yet been clarified. The purpose of the present study is to examine the growth pattern of the dentinal formation and evaluate the relative contributions of both genetic and environmental factors to longitudinal dentin formation by the use of the partial diallel cross-genetic approach, the time-marking method and a computerized image measurement system. The following results were obtained: 1) the onset of dentinal formation in the second molar was on the 6-9th day after birth, while that of the third molar was on the 9-12th day after birth; 2) the best fitted growth curve of the dentinal formation for the first molar was the fifth order polynomial equation, while for the second molar the fourth order polynomial equation was best, and for the third molar the third order polynomial equation was best; 3) maternal effect strongly influenced the second and the third molar dentin growth at an early stage of postnatal growth.

Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it.