18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

Recognition of the beta-2 microglobulin-B molecule by a CTL clone

In this report we offer evidence that the beta-2 microglobulin-B (beta 2M-B) molecule is recognized by a cytotoxic T lymphocyte (CTL). Production of GA5, a CTL clone reactive against a membrane antigen with the same strain distribution pattern as beta 2M-B, is described. This clone lysed syngeneic target cells in which native beta 2M-A molecules had been exchanged with beta 2M-B molecules by incubating the cells in serum from beta 2M-B-positive mouse strains. Conversely, the CTL clone GA5 failed to lyse its specific target when beta 2M-B molecules had been exchanged with beta 2M-A molecules by incubating the cells in serum from beta 2M-A-positive, beta 2M-B-negative mouse strains. Strain combinations were chosen so as to limit reactivity to beta 2M and to preclude reactivity to H-3 antigens, thus indicating CTL clone GA5 to be reacting specifically with beta 2M-B.     

Additional mapping of mouse chromosome 2 genes

The purpose of this work was to elucidate the genetic fine structure of the central portion of mouse chromosome (Chr) 2. Seven Chr 2 congenic mouse strains [B10.PA(L)-pa we un a ^t , B10.PA(L)-pa A ^w , B10.PA(L)-we un a ^t , B10.PA(J)-pa a, B10.FS-we A ^w , B10.C-we A ^w , and B10.YBR-a] were produced. Breeding studies were carried out using strains B10.PA(L)-pa we un a ^t and B10.LP-H-13 ^b to accurately determine the recombination frequencies between marker genes pa and we (1.9%±0.3), we and un (8.8%±0.5), and un and a ^t (4.5%±0.4) of strain B10.PA(L)-pa we un a ^t . These strains and other Chr 2 congenic strains were typed for immunologically defined loci using monoclonal antibody (mAb) C23 reactive with the gene product of B2m ^b T-lymphocyte clone C1 reactive with the gene product of H-3 ^a and H-3 ^c , and lymphocyte clone H1.8 reactive with the gene product of Hd-1 ^a . B2m and H-3 typing located a recombinational event separating [pa B2m H-3] from we (the order of bracketed genes is not known). Hd-1 typing indicated that Hd-1 maps distal to [H-42, H-44] and proximal to un. The gene order [pa, B2m, H-3], we, [H-42, H-45], Hd-1, un, H-13, a ^t , with H-44 mapping centromeric to Hd-1, is indicated by the data. Address correspondence and offprint requests to: R. J. Graff.     

CTL and serologically defined antigens of B2m,H-3 region

The antigens of the B2m,H-3 region of 13 chromosome 2 congenic strains and seven inbred strains have been studied by using CML and serologic techniques. Nine patterns of cross-reactivity have been defined by CML assays. These results are in agreement with an extend previously described cross-reactivity studies. The reactivities of three monoclonal antibodies previously thought to be reacting with B2M-B are shown to differ: Ly-m11 and J-5 react with cells of strain B10-pa,at and clone 23 does not. Two H-3 region loci are hypothesized on the basis of CML and serologic activity: B2m and H-3. The CTL responses to the B2M antigens are H-2K restricted; the CTL responses to H-3 antigens are H-2D restricted. The restriction of the response to the H-3 antigen requires effector-target identity of the H-2D molecule but not the B2M molecule of the class I antigen. These loci have been separated by recombination from H-42 in the production of the congenic strain B10.FS-a. A gene order of B2m, H-3, H-42 is suggested.     

Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids

Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.     

The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C

Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.     

Separation of 5'-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography

We have developed computer programs for characterization of ligand-binding systems in terms of continuous affinity distributions of arbitrary shape based on a numerical finite difference method. This method provides an excellent initial estimate of the affinity distribution, which can be further refined by means of nonlinear least-squares curve fitting. The method has been extensively tested for several cases including receptor heterogeneity, cooperativity, and for several examples of experimental design (e.g., ligand concentrations), and various levels of random and systematic experimental errors. The results provide a guide to experimental design, and indicate limits to the resolution obtained by ligand-binding studies, irrespective of the method of analysis.     

The alleles of theH-13 locus

Seven congenic strains differing from C57BL/10Sn at theH-13 locus have been produced which define fourH-13 alleles. Isografting, exchanging of grafts between sublines, F(1) testing, and linkage testing demonstrate the presence of additionalH genes in four of these strains. The medial survival times (MSTs) of skin grafts fromH-13(a) to unimmunizedH-13(b) recipients ranged from 69 to 83 days. Rejection across all other barriers was extremely weak with most MSTs being > 100 days. Preinjection of donor strain thymocytes caused accelerated rejection of skin grafts fromH-13(a) toH-13(b) mice, but had only minimal effect on skin grafts across other barriers. Rejection ofH-13 incompatible grafts was significantly stronger when the donor and host areH-3(a) than when they wereH-3(b).     

Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H₂O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pK_a = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.