Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae.

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. Porphyromonas gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes, 40 and 43 kDa, in the cell. The 43-kDa and 40-kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40-kDa Mfa3 alone. Furthermore, the 40-kDa Mfa3 started with the Ala(44) residue of the deduced amino acid sequence, indicating that the N-terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno-electron microscopy revealed that Mfa3 localized at the tip of the fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in the absence of other accessory proteins, PGN0290 and PGN0291, in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto-aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1(-) , FimA(-) ). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis.

Radioimmunoassay for somatostatin receptor type 2.

OBJECTIVE: To develop radioimmunoassay for somatostatin receptor type 2 (SSTR2) and search for its presence in certain rat tissues. METHODS: Anti-SSTR2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR2 with bovine serum albumin. Radioiodination of SSTR2 was performed by chloramin T method followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with SSTR1, SSTR3, SSTR4, SSTR5, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. SSTR2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue SSTR2 was about 89 %, and the intra-assay and inter-assay variations were 4.9 % and 7.8 %, respectively. SSTR2 was found in the hypothalamus, cerebrum, cerebellum, pituitary, stomach and testis. CONCLUSIONS: These data suggest that this assay system is suitable for the estimation of SSTR2 in the tissues.

Radioimmunoassay for hypocretin-2.

OBJECTIVE: To develop radioimmunoassay for hypocretin-2 (Hcrt-2). And search for its presence in certain rat tissues. METHODS: Anti-Hcrt-2 serum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic Hcrt-2 with bovine serum albumin. Radioiodination of Hcrt-2 was performed by chloramine T method, followed by purification of radoiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not cross react with hypocretin-2, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. Hcrt-2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue Hcrt-2 was about 85 % and the intra-assay and inter-assay variation were 5.6 % and 8.0 %, respectively. Hcrt-2 was found in the hypothalamus, cerebrum, brain stem and testes. CONCLUSIONS: The obtained data suggest that the assay system developed is suitable to measure Hcrt-2 in tissues and that Hcrt-2 is mainly found in the hypothalamus.

Radioimmunoassay for orexin A.

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.

Distribution of thyrotropin releasing hormone receptor type 2 in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of thyrotropin releasing hormone receptor (TRHR) type 2 in rats by immunohistochemical method. METHODS: TRHR type 2 was identified immunohistochemically in the rat tissues using specific anti-TRHR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic TRHR type 2 (5-23) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: TRHR type 2 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, Auerbach's and Meissner's nervous branch of the stomach, small intestine and colon, retina amd testis. Significant stain was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic TRHR type 2(5-23) or anterior pituitary homogenates, no significant stain of anterior pituitary was detected. CONCLUSIONS: These findings suggest that TRHR type 2 is widely distributed and that the method used is valuable in studying the distribution of TRHR type 2 in rats.

Nociceptin stimulates thyrotropin secretion in rats.

Effects of nociceptin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion in rats were studied. Nociceptin (150 microgram/kg) was injected intravenously and rats were serially decapitated after the injection. The effects of nociceptin on TRH release from the hypothalamus and TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormones were measured by individual radioimmunoassays. TSH was determined by enzyme immunoassay. TRH contents in the hypothalamus decreased significantly after nociceptin injection, whereas plasma TRH concentrations showed no changes. Plasma TSH concentrations increased significantly in a dose-related manner. The TRH release from the hypothalamus was enhanced significantly in a dose-related manner with the addition of nociceptin. The TSH release from the anterior pituitary in vitro was not affected by the addition of nociceptin. The plasma thyroxine and 3,3',5-triiodothyronine levels did not change significantly after nociceptin administration. The inactivation of TRH by plasma or hypothalamus in vitro after nociceptin injection did not differ from that of controls. The findings suggest that nociceptin acts on the hypothalamus to stimulate TRH and TSH secretion.