Complete determination of disulfide bonds localized within the short consensus repeat units of decay accelerating factor (CD55 antigen).

Decay accelerating factor (DAF) has 4 SCR (short consensus repeat) units. Each SCR unit consists of approx. 60 amino acids characterized by having four conserved cysteine residues and several other highly conserved residues which include proline, tryptophan, tyrosine/phenylalanine and glycine. To determine the disulfide-bonding pattern, we used the urine form of DAF. After thermolysin and trypsin digestion, we isolated seven disulfide-linked peptides by HPLC purification. Because all of the cysteine residues are disulfide-bonded, DAF should contain eight disulfide bonds. After subtilisin and trypsin digestion, we isolated the eighth disulfide-bonded peptides by HPLC purification. From sequence analyses of these peptides, we could identify all disulfide bonds in the 4 SCR units of DAF as being between the first and the third and between the second and the fourth half-cystines within each SCR unit.

Determination of the active site of CD59 with synthetic peptides.

CD59 inhibits the formation of membrane attack complex (MAC) of human complement by binding to C8 and C9 in the nascent membrane attack complex and inhibiting C9 binding to C8 in C5b-8 and C9 polymerization. Considering five disulfide bridges of CD59, we divided the molecule into two portions and synthesized the two peptides. One represented an amino-terminal half, P1-41, consisting of residues 1-41, while another represented a carboxyl-terminal half, P42-77, consisting of residues 42-77. P1-41 inhibited the MAC formation much more strongly than P42-77, indicating that the amino-terminal half contained the active site. We further synthesized P4-18 that consisted of residues 4-18 and P19-41 that consisted of residues 19-41. The activity of P4-18 was less than that of P19-41. Surprisingly, P19-41 showed higher activity than P1-41 and was comparable to urine CD59. Residues 19-41 were further divided into two portions: P20-25 which consisted of residues 20-25 and P27-38 which consisted of residues 27-38. Although their activities were significantly less than the activity of P19-41, P27-38 showed higher activity than P20-25. Residues 27-38 were further divided into three portions: P27-32 which consisted of residues 27-32, P30-34 which consisted of residues 30-34 and P33-38 which consisted of residues 33-38. When these peptides were assayed for the activities, all of them showed significant activities, even though they needed 10-fold more concentrations than P19-41. These data suggest that the portion made up of residues 27-38 is the active site constituting the binding site to C8 and C9.

Detection and identification of Entamoeba gingivalis by specific amplification of rRNA gene.

A pair of oligonucleotide primers were designed from the nucleotide sequence of the gene encoding the small subunit ribosomal RNA (SrRNA) of the oral protozoan parasite Entamoeba gingivalis. The primers amplified a 1.4-kb DNA fragment by polymerase chain reaction and were specific for Entamoeba gingivalis but not for other protozoa, oral protists and bacteria, or human leukocytes. With this method, the DNA from as few as 30 cells of Entamoeba gingivalis could be detected. These results suggest that this approach is applicable to the detection and identification of Entamoeba gingivalis in the human oral cavity.

Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction.

A polymerase chain reaction (PCR) protocol was developed for specific detection of Trichomonas tenax by using a pair of primers designed for its 18S rRNA gene. The detection was specific for T. tenax, since no amplification was detected with DNAs from Trichomonas vaginalis, which belongs to the same genus as T. tenax, in addition to various species of oral protists, fungi and bacteria, and human leukocytes. This method had a detection limit of 100 fg for T. tenax genomic DNA and could detect T. tenax cells in dental plaque at a concentration of as low as 5 cells per PCR mixture. Direct detection from clinical dental plaque samples was also possible; therefore, the present PCR procedure could provide a simple and rapid detection method of T. tenax in dental plaque.

Extracranial chondroma of the skull base.

We report a case of an extracranial chondroma of the skull base. This tumor occupied mainly the skull base, the nasopharynx and the parapharyngeal space. Magnetic resonance imaging was effective in diagnosis and determining the extent of the lesion. Total extirpation of the lesion was performed via an external cervical approach. A survey of the available literature revealed only three similar cases treated to date.

Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.

The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.