Novel antibody assessment method for microbial compositional alteration in the oral cavity

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.     

A staphylococcal coagglutination test for detecting and serogrouping Legionella pneumophila

For detection of Legionella pneumophila and determination of its serogroup, the modified staphylococcal coagglutination test was studied in detail. Cross-reactions in serum-agglutination tests were observed among serogroups, but immunoglobulin-coated staphylococcal cells could detect L. pneumophila with high sensitivity (a cell concentration with an absorbance of 0.008 at 660 nm could be detected) and serogroups could be determined without cross-reactions. Moreover the coexistence of other bacteria did not affect the results of the test. These results suggest that the staphylococcal coagglutination test is useful for detection and identification of Legionella, especially in environmental samples, and for serogrouping of isolates of L. pneumophila from clinical specimens.     

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.     

Targeting of Chrolamphenicol Acetyltransferase to Human Immunodeficiency Virus Particles via Vpr and Vpx

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N-terminus of HIV-1 Vpr, HIV-2 Vpr, or HIV-2 Vpx was incorporated into mature virions in a type-selective manner. By using chimeric proteins between HIV-1 Vpr and HIV-2 Vpx, we found that the N-terminal side of these proteins was mainly important for type-selective virion incorporation. The C-terminal arginine-rich region of HIV-1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV-2 Vpr and HIV-2 Vpx had no such activity. This region of HIV-1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.