Muscarinic acetylcholine receptors in rat gastric mucosa

Summary The muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.     

Cell-mediated cytotoxicity against allogeneic mutant H-2 antigens: restricted and nonrestricted responses

Two "gain and loss" type mutations of the H-2D region, the H- 2bm13 and H- 2bm14 , resulted in the expression of noncross-reactive CML determinants that are unique to each mutation, the Dbm13 gains and Dbm14 gains, respectively. According to the results of direct cytolytic and competitive inhibition assays of in vitro induced primary cytotoxic T lymphocytes, allogeneic responses specific for Dbm13 gains are generated by responders bearing the H-2b ( KbIbDb ) haplotype, but not by responders bearing the H- 2bm14 ( KbIbDbm14 ), KbIbDd , KbIbDk , or KbIb / qDq haplotype. Responses by the non-H-2b responders against Dbm13 are limited to those determinants shared by the Dbm13 and Db molecules. Because congenic mice differing only at the H-2D region are either responsive or nonresponsive to Dbm13 gains, the responsiveness is controlled by gene(s) in the H-2D region. F1 hybrid offspring of responsive (H-2b) and nonresponsive (non-H-2b) parents are invariably responsive, indicating genetic dominance of the responsiveness. In contrast to the response against Dbm13 gains, cytotoxicity specific for Dbm14 gains is generated by responders bearing the H-2b, H- 2bm13 , KbIbDd , KbIbDk , or KbIb / qDq haplotype. These data indicate the existence of two types of allogeneic MHC determinants; one, represented by Dbm14 gains, is the classic type capable of eliciting CML responses in mice of a wide range of H-2 haplotypes, whereas the other, exemplified by Dbm13 gains, elicits CTL responses only in mice of a few related haplotypes. It is proposed that recognition of Dbm13 gains is restricted by structures shared by Db and Dbm13 but missing from other D (or L, R, etc.) molecules, such as Dbm14 , Dd, Dk, and Dq. Availability of various restricting structures in self MHC molecules may thus influence the alloreactive CTL repertoire.     

Steady-state kinetics and spectral properties of Corynebacterium sarcosine oxidase

The overall reaction kinetics of Corynebacterium sarcosine oxidase were investigated and the reaction was shown to follow a ping-pong, bi-bi mechanism with two substrates, sarcosine and molecular oxygen. Sarcosine analogs, such as acetate, propionate and methoxyacetate, were competitive inhibitors of the reaction. Acetate caused characteristic alterations in optical and circular dichroic spectra, indicating that the microenvironment of the substrate-binding region of the enzyme increased in hydrophobicity on binding with the substrate analog. The dissociation constants of the analogs calculated from the spectral changes were in agreement with the kinetic inhibition constants. Inorganic metallic ions were also inhibitory. Of interest was the finding that the inhibition by Hg2+ was proportional to the square of its concentration, which suggests that at least two sulfhydryl groups are related to the catalytic activity of the enzyme.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.