Arabino-mycolates derived from cell-wall skeleton of Mycobacterium bovis BCG as a prominent structure for recognition by host immunity.

Arabino-mycolates are components of the cell-wall skeleton of Mycobacterium bovis BCG (BCG-CWS). It is known that synthesized arabinomycolates induce the production of tumor necrosis factor alpha (TNF-α) in murine macrophage cell lines at an intensity similar to that of BCG-CWS. However the immunological activity of natural arabino-mycolates isolated from BCG has not been investigated, probably due to the complexity of the molecule. In this paper, we investigated the immunostimulatory activity of arabino-mycolates isolated from BCG-CWS by acid hydrolysis. Arabino-mycolates obtained by acid hydrolysis from the originally prepared CWS (SMP-105) of M. bovis BCG Tokyo 172 strain consisted mainly of mono-arabinose mono-mycolate, penta-arabinose tetra-mycolate and hexa-arabinose tetramycolate fractions. Arabino-mycolates significantly induced TNF-α production with an intensity comparable to that of CWS and enhanced delayed type hypersensitivity (DTH) reactions against inactivated tumor cells. Arabino-mycolates-induced TNF-α production was completely dependent on TLR2 and MyD88 pathways. These findings indicate that isolated natural arabino-mycolates possess potent adjuvant immunostimulatory activity.

Bound thrombin-induced upregulation of myosin heavy chain isoform, SMemb messenger RNA expression in cultured rabbit vascular smooth muscle cells.

To investigate whether bound thrombin can induce modulation of SMemb expression in vascular smooth muscle (VSM) cells, messenger RNA (mRNA) expression was measured by in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) in cultured rabbit aortic VSM cells. To test the concentration- and time-dependent effect of bound thrombin on the expression of SMemb, confluent VSM cells were incubated for 48 h in 10% FBS-DMEM containing 0, 3, 10 and 30 units/ml of bound thrombin. In addition, the confluent VSM cells were incubated for 6, 12, 24 and 48 h in 10% FBS-DMEM containing 10 units/ml of bound thrombin. Consequently, bound thrombin significantly increased SMemb mRNA in a concentration- and time-dependent manner. When compared with the effect of rabbit fibrinogen (10 microg/ml) and native thrombin (10 units/ml), SMemb mRNA was significantly increased by bound thrombin and was slightly increased by native thrombin, but not by fibrinogen. Other myosin heavy chain (MHC) isoform (SM1 and SM2) mRNA expressions were not changed by fibrinogen, native thrombin or bound thrombin. ISH revealed that there was no significant difference in the expression of MHC mRNAs among fibrinogen, native thrombin or bound thrombin. Western blot analysis demonstrated that the SMemb protein level was significantly increased by 2.5-fold by bound thrombin. When the clot-forming activities in cultured medium containing native thrombin or bound thrombin were measured from 0.5 to 48 h, the activity of bound thrombin declined more slowly than that of native thrombin. In conclusion, bound thrombin could upregulate the expression of SMemb mRNA and protein in cultured VSM cells and the activity of bound thrombin was maintained for longer than that of native thrombin in culture medium.

Correlation between telomerase activity and telomeric-repeat binding factors in gastric cancer.

Telomeres of a specific length are essential for continuous cell proliferation. The length of telomeres must be maintained by telomerase action and the telomeric DNA-repeat binding protein must be protected. Therefore, there seems to be a relationship between cell immortality due to telomerase activity and telomeric DNA-repeat binding protein. We examined telomerase activity and the expression of telomeric-repeat binding factor 1 and 2 (TRF1 and TRF2) in gastric cancer. Telomerase activity was semi-quantified using the f-TRAP technique in 53 cancerous and non-cancerous gastric tissue specimens. TRF1 and TRF2 were also studied using an immunohistochemical method to determine the frequency of these factors in cell nuclei. Telomerase activity was observed in 79.2% of the cancerous tissue and in 39.6% of the non-cancerous tissue. The average semi-quantitative values for telomerase activity were 67.3 total product generated (TPG) unit/microg protein in cancerous tissue and 6.0 TPG unit/microg protein in non-cancerous tissue. Moreover, T0/1 tumor had the same incidence of telomerase activity as T2 or deeper tumors. These results indicated that the activation of telomerase begins at an early stage of carcinogenesis. TRF1 and TRF2 were detected in 45.1% and 42.9% of the cancerous tissue and in 70.6% and 65.6% of the non-cancerous tissue, respectively. In addition, low positive staining ratios were found for TRF1 and TRF2 when cancer had more deeply invaded. However, telomerase activity did not correlate with either TRF1 or TRF2. These findings suggest that optimal conditions for efficient telomerase are produced as cancer progresses, via suppression of TRFs.

Isolation of bound thrombin consisting of thrombin and fibrin N-terminal fragment from clot lysate.

It has been reported that thrombin is liberated from fibrin clots by the action of fibrinolytic enzymes. It has also been reported that the liberated thrombin complexes with fibrin fragment E or (DD)E, which are denoted as bound thrombin. However, bound thrombin has not been isolated from clot lysate, and the structural characteristics of isolated bound thrombin have not been specified. In this study, we attempted to isolate the bound thrombin from clot lysate and to clarify its structural features. Rabbit fibrinogen was clotted with bovine thrombin, and clot lysate was prepared with urokinase. The bound thrombin was isolated from clot lysate by serial chromatography using a Sepharose 4B column immobilizing an anti-bovine thrombin antibody and a Sepharose 4B column immobilizing an anti-rabbit fibrinogen antibody. SDS-PAGE under unreduced conditions demonstrated that there were two different protein bands in the isolated bound thrombin. On a C4 reverse-phase HPLC, the bound thrombin from clot lysate was resolved by 4 M urea into alpha-thrombin and a fibrin fragment, the N-terminal regions of which were identified as alpha-, beta- and gamma-chains. Thus, in the bound thrombin, thrombin molecule would bind to rabbit fibrin fragment consisting of N-terminal central domain.

Analysis of IgE turnover in non-sensitized and sensitized rats.

BACKGROUND: Although the levels of immunoglobulin E (IgE) in the circulating blood are often elevated in patients with allergic diseases, such levels cannot always be considered as pathognomonic signs of allergy. The induction of allergic reactions in the tissue was inferred to be related to the amount of IgE passing through the vascular wall. AIMS: We attempted to clarify which compartment, the intravascular or extravascular, plays an important role in the regulation of the turnover of rat IgE. METHODS: The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. RESULTS: The transfer rate constants from the central to tissue compartment (Kct) were larger than those from the tissue to central compartment (Ktc) irrespective of the sensitized state. The value of the distribution volume of the tissue compartment (Vt) was larger than that of the distribution volume of the central compartment (Vc) irrespective of the sensitized state. CONCLUSIONS: These Findings suggest that the short half-life of rat IgE in the circulation could be attributable to the distribution of IgE from the intravascular to the extravascular compartment.

Expression of p16, nm23-H1, E-cadherin, and CD44 gene products and their significance in nasopharyngeal carcinoma.

OBJECTIVE: The present study was aimed to determine whether p16/MTS1, nm23-H1, E-cadherin, and CD44 proteins were expressed in nasopharyngeal carcinoma (NPC) and whether those expressions were pathologically significant in the progress of NPC. METHOD: We examined non-cancerous nasopharyngeal mucosa (20 cases) and NPC (80 cases) using immunohistochemistry with six different types of monoclonal antibodies against p16, nm23-H1, E-cadherin, CD44H, CD44v3, and CD44v6 proteins. RESULTS: The results showed that 1) the rates of positive p16 protein expression and of preserved E-cadherin protein expression in NPC were significantly lower than those in non-cancerous tissue (P <.01); 2) no significant difference in the rate of positive expression of nm23-H1, CD44H, CD44v3, and CD44v6 proteins were observed between non-cancerous nasopharyngeal mucosa and NPC; 3) no significant difference in the expression of those proteins were found by respective correlation analyses of sex, stage, and size of primary tumor in NPC; and 4) no significant difference in the rates of positive expression of CD44H, CD44v3, and CD44v6 proteins were observed in NPC between with and without lymph node metastasis, indicating that those gene products did not correlate with lymph node metastasis in NPC. However, there were inverse correlations between the expression of p16, nm23-H1, or E-cadherin protein and lymph node metastasis (P <.05), indicating that the expression of p16, nm23-H1, and E-cadherin gene were related to the carcinogenesis and tumor progression of NPC. CONCLUSION: Detecting the expressions of those gene products may provide clinically valuable information for therapeutic strategy and for predicting the prognosis of patients with NPC.

[Effectiveness of Saiko-Keishi-To (TJ-10, a Kampo herbal medicine) for trigeminal neuralgia in rats].

It is known that Saiko-Keishi-To (TJ-10), a Kampo herbal medicine used for the treatment of epilepsy, shows a satisfactory curative effect even in the patients suffering from trigeminal neuralgia. To verify the effectiveness of TJ-10, Wistar rats with chronic neuralgia of the mandibular nerve were prepared and TJ-10 was administered to them for 4 weeks following the manifestation of pain in the mandibular region. The result reveals that the rise in the pain threshold in the mandibular region is more significant in the rats administered TJ-10 than in those in the control group. However, in the tail flick test, no significant change was observed in the pain threshold. These findings suggest that TJ-10 is effective for controlling the manifestation of pain in ligatured nerves, by local effect, not by general analgesic effect.

Conformational analysis of tandospirone in aqueous solution: lead evolution of potent dopamine D4 receptor ligands.

The significant contribution of folded conformation (2) of the anxiolytic tandospirone (1) in aqueous solution was verified by dynamic 1H NMR. A structurally rigid mimic of 2 was designed and synthesized to evaluate the implication of 2 towards neuroleptic receptor binding. The designed structures provided a new rigid scaffold for dopamine D4 ligands.

Fine needle aspiration of toxoplasmic lymphadenitis in an intramammary lymph node. A case report.

BACKGROUND: Cytologic findings of toxoplasmic lymphadenitis (TL) have been only sporadically reported. Intramammary lymph node is an extremely rare site for TL. CASE: A 47-year-old, healthy, female presented with a breast tumor, which was aspirated. The cytomorphologic features were interpreted as suggestive of TL. Histopathology of the excisional biopsy specimen and subsequent serologic examination confirmed the diagnosis. CONCLUSION: We obtained several characteristic findings in aspiration of TL. Of these, epithelioid cell clusters and monocytoid cells were the most diagnostic.