Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37 degrees C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.     

Etodolac: effect on prostaglandin concentrations in gastric mucosa of rats

Etodolac is a structurally novel compound exhibiting potent analgesic and anti-inflammatory activity in laboratory animals and man, with excellent G. I. tolerance. Like other nonsteroidal anti-inflammatory drugs (NSAIDs) etodolac inhibits prostaglandin (PG) biosynthesis. In view of the cytoprotective role of PGE2, we have investigated in normal rats the effect of etodolac on the gastric mucosal concentration of PGE2 as well as of 6-keto-PGF1 alpha, the stable metabolite of prostacyclin; naproxen and piroxicam served as reference NSAIDs. The orally effective anti-inflammatory doses in the chronic arthritic rat model (3 mg/kg for etodolac and naproxen; 0.5 mg/kg for piroxicam), and their arbitrarily selected multiples of 10 were used. Rats were killed at 1, 2, 6 and 24 hr after single doses and the PG concentrations were measured by RIA. With the low dose, 2 and 6 hr after dosing, etodolac diminished the PGE2 concentration by 20-25% (vs control) while naproxen and piroxicam caused a fall of 53-65%; the difference between etodolac and the untreated control group is not statistically significant but the difference between etodolac and both piroxicam and naproxen is significant (p less than 0.001). At the high doses, the lowering in PGE2 was similar after all three drugs, i.e. about 70% at 1 and 2 hr; 50% at 6 hr, and 20-50% at 24 hr after dosing. Except for the consistently smaller reduction of concentrations after etodolac, the effects on 6-keto-PGF1 alpha concentration followed a similar pattern but the differences are not significant. The lack of the G.I. irritation of etodolac in rats and man at therapeutically effective doses may be attributed to the benefits of the relatively short-lived and slight decrease in gastric mucosal PGE2 concentrations found in this study.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Neurospora Trehalase and Its Structural Gene

We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.     

Phosphorylation of ribosomal and ribosome-associated proteins in isoproterenol-induced cardiac hypertrophy

Cardiac hypertrophy in adult rabbits was induced by subcutaneous injection of isoproterenol. The rate of [3H]leucine incorporation into acid insoluble material was increased and the extent of [32P]phosphate incorporation into several ribosomal proteins was altered. Specifically, a ribosomal protein with a molecular weight of 32,000 from the 40S ribosomal subunit showed a five-fold increase in phosphate incorporation in the hypertrophic heart whereas a protein with a molecular weight of 28,000 from the 60S subunit showed a four-fold decrease. Phosphorylation of ribosome-associated proteins, which could be removed from ribosomes with 0.72 M KCl, was also changed in the hypertrophic hearts. Six major phosphoproteins (with molecular weights 62,000, 49,000, 36,000, 30,000, 20,000 and 12,000) were detected in both the normal and the hypertrophic hearts. Phosphorylation of the 62 K and the 49 K protein was increased by two- and three-fold, respectively, in the hypertrophic hearts, whereas phosphorylation of the 36 K and the 30 K protein decreased by two-fold. The level of phosphorylation of the 20 K and the 12 K protein was not significantly changed in hypertrophic hearts.     

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H2 metabolism in Escherichia coli.

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2 (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium-Activated Proteolysis

The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium-activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000-200,000 and 110,000-120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton-insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calcium-activated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium-activated NF protease in neural tissues.