Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution

The inhibitory effect of a high external Ca²⁺ ([Ca²⁺]_o) on spontaneous transmitter release in a high K⁺ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca²⁺ ([Ca²⁺]_i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca²⁺]_i; increasing [Ca²⁺]_o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K⁺ concentration (7–15 mM), their facilitatory effects on m.e.p.p. frequency decreased, disappeared and eventually reversed to depressant actions. Since a rise in the external K⁺ concentration would increase the steady state level of [Ca²⁺]_i, it is suggested that when the [Ca²⁺]_i is preset at a high level, manipulations so as to further increase [Ca²⁺]_i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca²⁺]_i.     

Toll-like receptor 9 is expressed on follicle-associated epithelia containing M cells in swine Peyer's patches

The precise distribution and expression of Toll-like receptor (TLR) 9 in gut-associated lymphoid tissues (GALTs) has not been elucidated. In this study, we investigated the expression pattern of TLR9 in adult and neonatal swine GALTs by real-time quantitative PCR, western blot, confocal laser microscopy and flow cytometric analysis. The swine TLR9 gene was preferentially expressed in adult Peyer's patches (Pps) and mesenteric lymph nodes (MLNs), which contained approximately three times higher TLR9 than the spleen. Other tissues exhibited only weak expression of TLR9. In neonatal swine, elevated expression of TLR9 was detected only in MLNs. We firstly showed that highly expressive (TLR9(+)) cells were formed in Pps and MLNs. In addition, TLR9(+) cells were present not only in immune cells such as dendritic cells and B cells but also in follicle-associated epithelia (FAE) including membranous cells (M cells) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses.     

The distinction between Burkitt lymphoma and diffuse large B-Cell lymphoma with c-myc rearrangement.

To compare immunophenotypic and molecular features between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) with c-myc rearrangements (c-mycR DLBCL), we analyzed 18 cases of B-cell non-Hodgkin's lymphoma with c-mycR that were confirmed by chromosomal and/or Southern blotting analyses. The cases were histologically classified into 10 BLs and five DLBCLs. The remaining three cases could not be classified because of suboptimal quality of the surgical materials. BLs were from five adults and five children, whereas all DLBCLs were from adults. BLs were positive for CD20 (10/10 cases examined), CD10 (9/10), Bcl-2 (1/9), and Bcl-6 (10/10), whereas they were negative for CD3 (0/10) and EBV (0/8), by Epstein-Barr virus (EBV) EBER-1 RNA in situ hybridization. c-MycR DLBCLs were positive for CD20 (5/5), CD10 (2/5), Bcl-2 (3/4), and Bcl-6 (4/4), whereas none of them were positive for CD3 and EBV. A mean of MIB-1 index (MIB-1+ cells/neoplastic cells, %) of BLs (98.1%) was higher than that of c-mycR DLBCLs (66.3%; P <.0001). Somatic mutation of immunoglobulin heavy-chain gene variable region (VH gene) in BLs (four cases) ranged from 0.7 to 4.9% with an average value of 2.3%, whereas those in DLBCLs (three cases) from 8.2 to 32.0% with an average value of 17.0%. It is, therefore, concluded that a growth fraction of nearly 100%, as well as a monotonous proliferation of medium-sized cells and c-myc(R), should be of value in the diagnosis of BL, which is probably different from c-myc(R) DLBCL. In addition, CD10+, Bcl-2-, and low frequency of mutation of the VH gene could be helpful for the histologic distinction of BL from (c-mycR) DLBCL.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Saliva-binding region of Streptococcus mutans surface protein antigen.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.     

The ultrasound examination of the deep vein thrombosis

Ultrasonography is very useful for detection of deep vein thrombosis. The purpose of this paper is to show a method for detecting them efficiently by high resolution transducer and color Doppler system. We examined patients in the supine and prone positions. To detect the venous flow easily and differentiate thrombi from simple venous dilatation, some maneuvers are useful; one is pushing the vein area using the transducer on examination, the second is breathing overload, and the last is so-called milking. We can find throombi in the external iliac or femoral veins of patients who have symptoms of lower leg swelling, however, we need to better detect venous thrombi in the lower leg in patients with a history of pulmonary embolism. Because deep venous thrombi are increasing, the role of ultrasound will expand in the future.