Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.     

Molecular analysis of virus-producing and non-producing clones derived from a defective SSPE virus Yamagata-1 strain.

Two virus clones were isolated from a defective SSPE virus, the Yamagata-1 strain, and designated as the YA and YF clones. The YA clone-infected cells produced neither cell-free virus nor cell-associated virus, whereas the YF clone-infected cells produced both cell-associated and cell-free virus. No difference of epitopes on structural proteins was observed between these two clones. Both clones had hemadsorption activity. Quantitation of structural protein by Western dot blots showed relatively a lower amount of M protein in the YA-infected cells than that in the YF-infected cells. The ratio, P plus M dicistronic/M monocistronic mRNA, in the YA-infected cells was about twice that in the YF-infected cells. Sequence analysis of cDNA corresponding to P plus M dicistronic mRNA revealed that the deduced M protein of the YF virus was smaller than that of the YA virus by five amino acids from the carboxy terminal. These results suggest that abundant production of P plus M dicistronic mRNA is responsible for the reduced amount of M protein in the non-productive YA clone.     

Tooth replacement in the teleost fish Prionurus microlepidotus Lacépède

Previous studies on tooth replacement in lower vertebrates have been plagued by a lack of common integrative approaches and methods, making it impossible to furnish a phylogenetic synthesis. This study is based on serial sections of the jaw of Prionurus microlepidotus. Each Toothgerm was characterized by its developmental stage and its position in the jaw. The relationship between the developmental stage of toothgerm and position in the jaw has been studied and expressed in several graphical illustrations. The following conclusions have been made: (1) The initiation of toothgerms in P. microlepidotus is governed by two Zahnreihen, which respectively initiate toothgerms on the lingual and labial side of the functioning teeth in an alternating pattern. (2) Therefore, functioning teeth in one locus are supplied by the alternate eruption of lingual and labial toothgerms. (3) Advancing of tooth replacement in each locus is independent of functioning teeth and their successors in adjacent loci. (4) The disorders of replacement patterns are caused by an alternated rate of eruption of successive toothgerms as a response to unusual shedding of the functioning teeth.     

Observations on Nonconverting Phage,c-n71, Obtained from a Nontoxigenic Strain of Clostridium botulinum Type C

A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinum type C, Stockholm, with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm, it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.     

Effects of pressure on the phase transition of bilayers in liposomes influence of cholesterol and α-tocopherol

The effects of presure on the gel-to-liquid crystalline phase transition temperature of dimyristoylphosphatidylcholine (DMPC) bilayers containing cholesterol, α-tocopherol and α-tocopheryl acetate were studied by fluorescence depolarizalion. The transition temperature of cholesterol mixtures ( > 7.5 mol%) was lower than that of 100% DMPC at atmospheric pressure, but it became higher than the latter on increase in pressure. The thermodynamic parameters of the transition (ΔV,ΔS,ΔH) were estimated and the functions of cholesterol and α-tocopherols in the bilayers are discussed.     

Reproductive features of the Russian vole in laboratory breeding.

Reproductive features of newly bred Russian voles (Microtus rossiaemeridionalis) as a laboratory animal were studied. This species is a copulatory ovulator, and most couples copulated 6 to 16 h after pairing. The gestation period varied from 18 to 22 days (mean +/- SD: 20.6 +/- 0.9, n = 72), and the average litter size was 4.6 +/- 1.9 (n = 125). Compared with the litter size at the first parturition (3.6 +/- 1.6, n = 72), the litter size in the subsequent parturitions increased to 5.9 +/- 1.4 (n = 53). The animals exhibited postpartum estrus, and repeated pregnancy accompanied with suckling pups and parturition continuously in the laboratory condition unlike other vole species. In view of their complex stomach and good proliferation, the Russian voles were evaluated as a good laboratory animal, especially as a model animal for ruminant studies.     

Hydrophobicity of the cell surface and drug susceptibility of Escherichia coli cultivated at high pressures up to 30 MPa

Summary Escherichia coli was cultivated under hydrostatic pressures up to 30 MPa (300 bar) and then partitioned between an aqueous phase (physiological saline) and oil phase (n-hexadecane). The partition coefficients were used as measures of hydrophobicity of the surface of the cells and correlated with the susceptibility to an antimicrobial agent (dodecylpyridinium iodide). This agent is lethal to the cells and the effect of pressure on its concentration for a lethal effect on E. coli was determined. A good correlation was found between the hydrophobicity of the cells and their death rate on treatment with this reagent.