Gene expression and phenotypic traits of Aggregatibacter actinomycetemcomitans in response to environmental changes.

BACKGROUND AND OBJECTIVE: Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. MATERIAL AND METHODS: Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. RESULTS: The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apaH, omp29, cagE and cdtB) and in adhesion to extracellular matrix protein (emaA). CONCLUSION: Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential.

A phase diagram of the binding of mismatched duplex DNAs to hydroxyapatite.

The hydroxyapatite binding properties of imperfectly base-paired DNA have been investigated and summarized in a phase diagram. This diagram defines the conditions at which thermal elution results in both denaturation and desorption of mismatched duplex DNA from hydroxyapatite.

Conditional gene targeting and its application in the skin.

The knockout mouse is one of the most useful experimental animal systems in which to clarify functions of identified genes in vivo. Numerous knockout mice have been produced, and studies in these models have revealed new aspects in various biological fields. In fair cases, however, mice died during embryogenesis due to complete gene disruption, making it difficult to elucidate functions of given genes in adulthood. To avoid this, conditional gene targeting has been performed and shown to be effective on numerous cases. Here, we will overview established strategies for conditional gene targeting and present the results of our recent studies using the Cre/loxP system in the skin.

Purification of gizzard myosin light-chain phosphatase, and reversible changes in the ATPase and superprecipitation activities of actomyosin in the presence of purified preparation of myosin light-chain phosphatase and kinase.

Sepharose 4B conjugated with phosphorylated myosin light chains was used in affinity chromatography of a partially purified preparation of gizzard myosin light-chain phosphatase (MLCP) (Onishi et al. (1979) J. Biochem. 86, 1283-1290). The MLCP preparation thus purified contained, according to SDS gel electrophoresis, three components of 67,000 (67 K), 54,000 (54 K), 34,000 (34 K) daltons. In an accompanying report, Uchiwa et al. (J. Biochem. 91, 273-282 (1982)) described the purification of gizzard myosin light-chain kinase, which consisted of two subunits; 130 K and 17 K daltons. Using the purified preparations of MLCP and MLCK, it was demonstrated a) that reversible changes in the ATPase and superprecipitation activities occur as myosin light chains are enzymatically phosphorylated and dephosphorylated, and b) that addition of a very low concentration of Ca2+ and its removal cause reversible changes in the turbidity of actomyosin suspensions as well as in the state of phosphorylation of myosin light chains only when MLCK and MLCP are both present. These results provide strong support for the proposal (see Ikebe et al. (1977) J. Biochem. 80, 299-302) that MLCK and MLCP play a key role in the Ca2+ regulation in gizzard.

Early-onset facioscapulohumeral muscular dystrophy: two case reports.

This report concerns two patients with facioscapulohumeral muscular dystrophy (FSHD) whose facial weakness began in infancy. In both patients, biopsied muscle histology showed mild myogenic changes accompanied by some regenerating and some small angular fibers, while endomysial inflammatory cellular infiltration was observed in Patient 1. The finding that our very young patients had muscle histopathological findings compatible with classical FSHD supports the previously expressed view that muscle histopathology is not related to either age or duration of the disease. Although Patient 2 was a sporadic case, both patients had the abnormal EcoRI DNA fragment detected by Southern blot analysis with probes p13E-11 and pFR-1, a finding compatible with FSHD. This indicates that gene analysis of sporadic cases must be as significant as that of familial cases. This report on patients with very early-onset and with common muscle histopathological and molecular genetic findings should contribute to widening the clinical spectrum of FSHD.

[Experience of gamma-detecting probe for the survey of sentinel node in gastroesophageal malignancies].

Gamma-detecting probe NAVIGATOR GPS (Autosuture Japan) was evaluated by using of 99mTc. Linearlity of counting in radioactivity was fairly good, but the sensitivity of the prove is so low, that sentinel node (SN) should contain the 3.7 x 10(-3) MBq (0.1 microCi) of 99mTc or more for the most effective use of it. And the count rate of the gamma-detecting probe was influenced by the distance and angle from the 99mTc source variously. It is important for the operator to know such characteristics of the gamma-detecting probe in order to obtain the correct SN judging.

Signaling transduction analysis in gingival epithelial cells after infection with Aggregatibacter actinomycetemcomitans.

Periodontal diseases result from the interaction of bacterial pathogens with the host's gingival tissue. Gingival epithelial cells are constantly challenged by microbial cells and respond by altering their transcription profiles, inducing the production of inflammatory mediators. Different transcription profiles are induced by oral bacteria and little is known about how the gingival epithelium responds after interaction with the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. In the present study, we examined the transcription of genes involved in signaling transduction pathways in gingival epithelial cells exposed to viable A. actinomycetemcomitans. Immortalized gingival epithelial cells (OBA-9) were infected with A. actinomycetemcomitans JP2 for 24 h and the transcription profile of genes encoding human signal transduction pathways was determined. Functional analysis of inflammatory mediators positively transcribed was performed by ELISA in culture supernatant and in gingival tissues. Fifteen of 84 genes on the array were over-expressed (P < 0.01) after 24 h of infection with viable A. actinomycetemcomitans. Over-expressed genes included those implicated in tissue remodeling and bone resorption, such as CSF2, genes encoding components of the LDL pathway, nuclear factor-κB-dependent genes and other cytokines. The ELISA data confirmed that granulocyte-macrophage colony-stimulating factor/colony-stimulating factor 2, tumor necrosis factor-α and intercellular adhesion molecule-1 were highly expressed by infected gingival cells when compared with control non-infected cells, and presented higher concentrations in tissues from patients with aggressive and chronic periodontitis than in tissues from healthy controls. The induction in epithelial cells of factors such as the pro-inflammatory cytokine CSF2, which is involved in osteoclastogenesis, may help to explain the outcomes of A. actinomycetemcomitans infection.

Adhesion and invasion to epithelial cells by fimA genotypes of Porphyromonas gingivalis.

Adhesion to and invasion of epithelial cells by the periodontopathogen Porphyromonas gingivalis is promoted by the major fimbriae, encoded by fimA. The microorganism can be classified in six genotypes, based on fimA sequence, and genotype II strains are more prevalent than others in periodontitis patients. This study aimed to determine the adhesive and invasive abilities on KB cells of different fimA allelic variants of P. gingivalis isolates. Twenty-two isolates and six reference strains representing the six fimA genotypes and non-typeable strains were screened for their adhesion and invasion abilities on KB cells, using standard methods. All strains were able to adhere and, except for one, to invade KB cells. However, these properties were not homogeneous among strains belonging to the same genotype. There was no correlation between adhesion and invasion efficiencies. Isolate KdII 865 (fimA genotype II) was the most invasive and the second most adhesive strain, whereas reference strain ATCC 33277 (fimA I) showed a low adhesion ability but was highly invasive. These data indicated that fimA genotypes of P. gingivalis are not related to the adhesion and invasion abilities on KB cells, suggesting that the increased prevalence and proportion of certain genotypes may be attributed to other characteristics besides FimA variation.

Channeled-substrate-planar structure Al(x)Ga(1-x) As lasers: an analytical waveguide study.

The waveguide mechanism in channeled-substrate-planar (CSP) structure (AlGa) As lasers is analyzed. The light leakage into the GaAs substrate outside the channel is shown to provide effective refractive index and loss differences, which function to stabilize the transverse mode in the junction plane. Observed light output vs current characteristics and mode patterns in the lasers justify the theoretical model.

Transverse-mode stabilized Ga(1-x)Al(x)As visible diode lasers.

Transverse-mode stabilized visible diode lasers in the 0.7-microm wavelength region are fabricated by growing a Ga(1-x)Al(x)As double heterostructure on a grooved GaAs substrate (channeled-substrate-planar structure). The diodes operate stably in the fundamental transverse mode and provide nonstigmatic laser beams. They can be collimated with a 0.5-1-mrad beam divergence using a simple graded-index fiber lens. Corresponding to single-longitudinal-mode operation at current levels above 1.1 times the threshold, a coherence length as long as 14 m is obtained in Michelson interference experiments.

Distribution of fimA genotypes of Porphyromonas gingivalis in subjects with various periodontal conditions.

Fimbria encoded by the gene fimA is considered one of the main factors in the colonization of the oral cavity by Porphyromonas gingivalis. Allelic variation in fimA led to the classification of strains of P. gingivalis into six genotypes. The occurrence of P. gingivalis was determined by polymerase chain reaction using 16S rRNA primers in 302 subgingival samples obtained from 102 Brazilian subjects exhibiting different periodontal conditions. Distribution of fimA genotypes was assessed in 146 P. gingivalis positive samples by polymerase chain reaction using primers pairs homologous to the different fimA genes. P. gingivalis was detected in 51 of 57 (89.4%) patients with periodontal attachment loss, in six of 20 gingivitis patients (30.0%) and in two of 25 (8.0%) subjects with a healthy periodontium. Variant type II was the only type detected in 53 sites (39.3%), distributed among 19 periodontitis patients (37.3%) and in one patient with no periodontal destruction. Type Ib was the second most prevalent genotype in periodontitis patients (19.6%). Genotype V was not detected in the studied population. Type IV was the most commonly type found among gingivitis patients, either alone or in combination with other genotypes. Multiple genotypes were detected in nine sites (6.1%). A fimA genotype was not identified in 26 sites (17.8%) of 146 sites positive for P. gingivalis, suggesting that other alleles of fimA not yet sequenced may be prevalent in this population. These data demonstrated that P. gingivalis type II strains followed by type Ib are more prevalent in periodontitis patients from a multiracial population in Brazil, suggesting an increased pathogenic potential of these types.

Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure.

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.