Autoradiographic localization of nicotinic receptor binding in rat brain using [3H]methylcarbamylcholine, a novel radioligand

Light microscopic autoradiography was used to visualize the neuroanatomical distribution of nicotinic receptors in rat brain using a novel radioligand, [3H]methylcarbamylcholine (MCC). Specific [3H]MCC binding to slide-mounted tissue sections of rat brain was saturable, reversible and of high affinity. Data analysis revealed a single population of [3H]MCC binding sites with a Kd value of 1.8 nM and Bmax of 20.1 fmol/mg protein. Nicotinic agonists and antagonists competed for [3H]MCC binding sites in slide-mounted brain sections with much greater potency than muscarinic drugs. The rat brain areas containing the highest densities of [3H]MCC binding were in thalamic regions, the medial habenular nucleus and the superior colliculus. Moderate densities of [3H]MCC binding were seen over the anterior cingulate cortex, the nucleus accumbens, the zona compacta of substantia nigra and ventral tegmental area. Low densities of [3H]MCC binding were found in most other brain regions. These data suggest that [3H]MCC selectively labels central nicotinic receptors and that these receptors are concentrated in the thalamus, the medial habenular nucleus and the superior colliculus of the rat brain.     

Mechanism of polymorphonuclear leukocytes accumulation examined using inhibitors of complement and arachidonic acid cascade in rats treated with OK-432

When the streptococcal preparation OK-432 was intraperitoneally injected for the treatment of carcinomatous peritonitis, antitumor polymorphonuclear leukocytes (PMNs) accumulated in the peritoneal cavity. We examined the mechanism of this PMN accumulation using an in vivo system in rats. FUT-175, EDTA and K76 inhibited C5a generation by OK-432 in vitro, but EGTA, prednisolone and inhibitors of arachidonic acid cascade did not. In in vivo experiments, EDTA, FUT-175, antirat C3 serum and K76 reduced the accumulation of PMNs onto filter membranes, when these reagents were reacted with OK-432 for 3 h through filter membranes placed on the turned rat peritoneum. EGTA failed to inhibit PMN accumulation. Prednisolone, indomethacin, OKY046 and AA861 inhibited PMN accumulation in a dose-dependent manner. These inhibitions of PMN accumulation were confirmed by histological examination. It was concluded that complement-derived chemotactic factor C5a generated by OK-432 induced PMN accumulation in association with chemotactic arachidonic acid metabolites.     

Direct molecular interaction of caveolin-3 with KCa1.1 channel in living HEK293 cell expression system

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca²⁺-activated K⁺ channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.     

Alteration of DNA primase activity by phosphorylation and de-phosphorylation of histone H1

To investigate the effect of histone H1 on DNA primase activity, partially purified DNA primase from mouse FM3A cells was used. It was found that histone H1 dose dependently inhibited DNA primase. Interestingly phosphorylation of histone H1 reduced the inhibitory activity of the histone. However, de-phosphorylation of the phosphorylated histone H1 resumed the inhibitory activity of DNA primase. These findings lead us to the assumption that phosphorylation and de-phosphorylation of histone may regulate the cell cycle by controlling DNA synthesis through reverse inhibition of DNA primase.     

Macrophage dependency of T-lymphocyte mitogenesis by Nocardia rubra cell-wall skeleton.

The mitogenic activity of the cell-wall skeleton (CWS) of Nocardia rubra on purified splenic T-cells (thymus-derived lymphocytes) was investigated. N. rubra CWS showed remarkable mitogenic activity on normal spleen cells of C57BL/6J mice at concentrations ranging from 10 to 100 microgram per milliliter of culture medium, while, on purified splenic T-cells, N. rubra CWS did not act as an mitogen at any concentration. However, mitogenic activity of N. rubra CWS on T-cells was restored if purified splenic T-cells was reconstituted with X-irradiated peritoneal exudate cells (macrophages). The above results suggest the necessity of macrophages for T-lymphocyte activation by N. rubra CWS as well as PHA-P or Con A.     

An approach to chemotaxonomy of the Asarum subgenus

A survey of chemical composition of 23 species of Asarum subgenus Heterotropa showed that the five groups could be distinguished on the basis of the presence or absence of asatone, phenol ethers and terpenes.     

Seminal Plasma and Semen Amyloids Enhance Cytomegalovirus Infection in Cell Culture

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131:1059–1071, 2007; N. R. Roan et al., Cell Host Microbe 10:541–550, 2011; F. Arnold et al., J. Virol. 86:1244–1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.