Presence Of Mycophenolic Acid Research Articles

Tight links between adenine and guanine nucleotide pools in mouse pancreatic islets: a study with mycophenolic acid.

Glucose metabolism in pancreatic B-cells leads to an increase in the ATP/ADP ratio that might participate in the regulation of insulin secretion. Good correlations have also been observed between guanine nucleotide levels in isolated pancreatic islets and insulin secretion. To assess whether guanine nucleotides have a specific role in stimulus-secretion coupling, their concentration should be modified selectively. This was attempted by culturing mouse islets overnight in the presence of mycophenolic acid (MPA), an inhibitor of GMP synthesis at the level of IMP dehydrogenase. The drug (25-50 microg/ml) did not affect the insulin content but decreased the GTP content of the islets and inhibited insulin secretion during subsequent incubation in the presence of 15 mM glucose. However, MPA also decreased the ATP/ADP ratio in the islets. The addition of guanine to the culture medium (to stimulate the salvage pathway of GTP synthesis) restored normal GTP levels, corrected the ATP/ADP ratio and partly prevented the inhibition of insulin release. In contrast, attempts to stimulate ATP synthesis specifically (by provision of adenine or adenosine) failed to reverse any of the effects of MPA. It is concluded that guanine and adenine nucleotide pools are tightly linked and cannot be specifically affected by MPA in pancreatic islet cells, probably because of the activity of nucleoside diphosphate kinase and because of the role of GTP in several reactions leading to adenine nucleotide generation. Contrary to previous claims, MPA is not an adequate tool for evaluating a specific role of guanine nucleotides in the control of insulin secretion.

Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNFa and TNFβ We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker ( Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52–56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at earlytimes after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term “viroceptor” to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

Mutations that confer resistance to mycophenolic acid and ribavirin on sindbis virus map to the nonstructural protein nsP1

SV MPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SV MPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SV MPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SV MPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenotype. SV MPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5′ terminus of the Sindbis virus mRNAs.

Vaccinia virus DNA ligase is nonessential for virus replication: Recovery of plasmids from virus-infected cells

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).

Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: Evidence that the gene is essential for virus growth

Vaccinia virus encodes a type I DNA topoisomerase whose function in virus replication is not known. To determine whether topoisomerase is required for growth of vaccinia in cell culture, we attempted to isolate null mutations in the topoisomerase gene through insertional mutagenesis. Plasmids containing mutant topoisomerase alleles were constructed by intragenic insertion of the Escherichia coli gpt gene. Recombinant viruses containing the gpt insertion were isolated by selection for growth in the presence of mycophenolic acid. Analysis of the genome structures of drug-resistant viruses revealed that in every case ( n = 22) both the wild-type and the gpt-inserted allele were present in viral DNA. We interpret the retention of the wild-type allele as indicative of the essential nature of the topoisomerese gene for vaccinia virus growth.

Restoration by xanthine of development of mouse embryos inhibited by mycophenolic acid.

Mouse embryos at the 1-cell stage were cultured in various concentrations of mycophenolic acid and xanthine. Mycophenolic acid at 10 and 2.5 micrograms/ml completely inhibited development to the morula and blastocyst stages, respectively. The addition of xanthine reversed the inhibitory effects of mycophenolic acid on the embryos but the reversal effect depended on the concentration of xanthine. Development was normal with a xanthine concentration of 25 micrograms/ml, even when an inhibitory concentration of mycophenolic acid was present. Embryo transfer experiments showed that the blastocysts formed in vitro in the presence of mycophenolic acid + xanthine could implant in foster mothers and develop normally to fetuses.

Mouse melanoma antigen. Its immunological properties, partial structure and genes

The immunological and chemical properties of melanoma antigens have been analysed by using C57BL/6 derived B16 melanoma cells. Cytotoxic T lymphocytes or helper T cells involved in the positive immune responses were easily induced by the antigen on the cell surface, whereas soluble melanoma antigens secreted into the culture medium selectively stimulate and induce antigen-specific suppressor T cells. Furthermore, the soluble antigen works as an inhibitor for the blocking of the cytotoxic T cell activity. Therefore, the soluble melanoma antigen stimulates the negative immune response in order for tumor cells to escape from immune system. By using two syngeneic monoclonal antibodies against the soluble melanoma antigens (M2590 and M562), we found that the melanoma antigen was composed of GM3 (NeuAc) in association of proteins. M2590 recognized GM3 carbohydrate moiety with cross-species specificities, while M562 reacts with the protein with species specific melanoma antigenic determinant. M2590 can react with the extracted GM3 from normal cells, not with normal cells expressing GM3 on the cell surface. Thus, this suggests that the tertially structure of GM3 on melanoma cells is different from that of normal cells. Finally, we succeeded in cloning of the genomic deoxyribonucleic acid (DNA) which contains genes encoding melanoma antigen with species specific determinant (M562). The B16-melanoma DNA cosmid libraries were constructed with the shuttle vector pCV103. They were infected into ED8767 E. coli, and then transfected into the human melanoma cell line P-36 by protoplast fusion with polyethylene glycol. The transfectants were selected in the presence of mycophenolic acid, stained with FITC-labeled M562, and selected by FACS. Total DNA was isolated from the transfectants, and the B16 genomic DNA clones were rescued by the in vitro packaging with lysogenic bacterial extracts. pD2-7 (34.8kb) reproducibly expressed M562 determinants in the p-36 human melanoma cells.

Hypoxanthine: guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae.

Yeast mutants lacking activity of the enzyme hypoxanthine:guanine phosphoribosyltransferase (H:G-PRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4%, of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regultory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1+. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1+. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt+. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.