On the multiplicity of platelet prostaglandin receptors: II. The use of N-0164 for distinguishing the loct of action for PGI2, PGD2, PGE2 and hydantoin analogs

The ω-chain variant analogs of prostacyclin (PGI₂) and PGD₂ in which the n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1)(1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI₂, but converts PGD₂ to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD₂ by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD₂, cyclohexyl-PGD₂, and BW-245C; with essentially no effect on PGI₂, cyclohexyl-PGI₂ and PGE₂ at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre (2,3), but supports the view that the anti-aggregatory effect of high doses of PGE₂ ($\text{EC}{}_{50}\text{=50μ}\text{M}$) is mediated by the PGI₂ receptor (4). The hydantoin acts at the platelet PGD₂ receptor.     

Evidence that neutral spingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg²⁺-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, α-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg²⁺-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.     

Extracellular enzymes of some additional fungi associated with mushroom culture

Twenty-four fungus isolates from the compost utilized in commercially growing Agaricus brunnescens were tested for their ability to produce extracellular enzymes involved in the degradation of cellulose, lignin and xylan, the major components of the straw of the compost. All 24 isolates were able to degrade carboxymethyl cellulose. Most were classified as weak or moderate producers of exo--glucanase. Twenty of the 24 were also able to hydrolyze filter paper, a crystalline cellulose. Nineteen of the 24 were able to hydrolyze xylan, a hemicellulose. The production of extracellular polyphenol oxidases was detected utilizing two tests; the blueing of alcoholic gum guaiacol, which indicates tyrosinase production, and the browning of malt extract-gallic acid agar, which indicates laccase production. Twenty produced tyrosinase, but only eight produced laccase. Agaricus brunnescens was also included in all of the tests. It produced exo--glucanase, hemicellulase, tyrosinase and lactase.     

Cobalt transport in nickel-resistant strains ofNeurospora crassa

Uptake of Co²⁺ by three nickel-resistant strains (Ni^R1, Ni^R2, and Ni^R3) ofNeurospora crassa that differed in resistance to Co²⁺ has been studied. Uptake was linear with Co²⁺ concentration (up to 1 mM), with time (up to 6 h), and with pH between 3 and 6. Uptake rates were in the order Ni^R2>Ni^R1>Ni^R3. In all strains, there was gradual increase in Co²⁺ uptake between 10° and 28°C, with a much sharper increase between 28° and 40°C. Metabolic inhibitors decreased Co²⁺ uptake partially in all strains, except for KF in Ni^R3. About 50–80 g Co²⁺/100 mg dry weight was surface bound. Ni²⁺, Zn²⁺, and Mn²⁺ competed with Co²⁺, the effects being strain specific. Mg²⁺ inhibited Co²⁺ uptake in all strains with preformed mycelia. In Ni^R1 and Ni^R2 only with young mycelia (40 h old) was Mg²⁺ inhibitory to Co²⁺ uptake,during growth in the presence of Co²⁺. The results suggested the presence of two transport systems for Co²⁺ in Ni^R1 and Ni^R2, only one of which was sensitive to Mg²⁺; in contrast, Ni^R3 had a single system, which was sensitive to Mg²⁺.     

Polyamines and protein synthesis: Studies in various polyamine-requiring mutants ofEscherichia coli

Summary DifferentEscherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. Thein vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutants MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.Dedicated to ProfessorLuis F. Leloir on the occasion of ths 70^th birthday.     

Characterization of glycerol nonutilizing and protoperithecial mutants ofNeurospora

Summary Mutants defective in polyol metabolism and/or in protoperithecial development were selected inNeurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed intoN. crassa to facilitate genetic analysis. One,glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, betweenad-1 andrib-1; the other,glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal toad-9. Another mutant,gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal toad-9.     

Calf development and mother-calf spatial relationships in Southern right whales

Changes in spatial relationships between mother and calf right whales (Eubalaena australis) from birth to the separation of yearling calves from their mothers were observed. During the first few weeks of a calf's life, mother and calf were within close proximity over 90% of the time, and mothers were responsible for maintenance of contact with their infants. Later calves strayed farther and initiated many more leaves and approaches than their quiescent mothers. The mothers still maintained contact with their infants. Just before migration away from the area, pair members resumed close proximity and leaves and approaches by both diminished. Yearling calves, returning to the area with their mothers after six months, stayed close to their mothers and few leaves and approaches by either pair member were recorded. The yearling calves were responsible for maintaining contact as the mothers left them more than approached them. This behaviour on the part of mothers probably contributed to weaning of yearlings and separation after a few weeks in the area.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis.

It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 mM), a potent inhibitor of the transamination reaction, it was shown that there exists an "insulin-sensitive" pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of alpha-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.     

Reduced intracellular content of methotrexate in an isolated MTX-resistant cell line of Nicotiana plumbaginifolia

Communicated by R. N. Beachy