Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.     

Selection of fatty acid auxotrophs from the oleaginous yeast Cryptococcus curvatus and production of cocoa butter equivalents in batch culture

Summary A search was carried out for mutants, defective in the conversion of stearic acid to oleic acid in effort to improve the quality of lipids produced by Cryptococcus curvatus ATCC, 20509. Mutants were selected as unsaturated fatty acid (Ufa) auxotrophs. After treatment of parent organism with Ethyl methanesulfonate (EMS), 11 oleate-requiring auxotrophs were isolated. Only 3 of them were real unsaturated fatty acid (Ufa) mutants, while the other 8 were designated as fatty acid synthetase (Fas) mutants. The amount of saturated fatty acid (SFA) was about 65.2 % in the lipids extracted from an Ufa mutant named UfaM3 and it was significantly higher than that of the wild-type (WT) (46.6 %) and similar to that of cocoa butter (60.4 %).     

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.     

Detoxification of ochratoxin A,a food contaminant: Prevention of growth of Aspergillus ochraceus and its production of ochratoxin A

The toxicity of ochratoxin A (OTA), a mycotoxin produced by fungi ofAspergillus orPenicillium genera is now well documented. Its nephrotoxicity, immunosuppression, teratogenicity, and carcinogenicity have been widely studied. Physical and biochemical methods have been studied to prevent these toxinogenicAspergillus andPenicillium from producing OTA, and/or to destroy the mycotoxin when already produced in a liquid or a solid medium. Repeated freezing at ? 20?C and thawing at + 26?C aleatory reduce OTA production in a liquid medium. Exposure to UV B for different periods of time is efficient in preventing OTA production in a liquid medium. Gamma-irradiation from 2 to 5 kGy gives good results in preventing the production of OTA or destroying it when already produced. Carboxypeptidase is very efficient at 5 units/50 ml in a liquid medium for cleaving the OTA already produced.     

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy

The metabolism of glucose was studied in Lactococcus lactis subsp. lactis CNRZ 125 by ¹³C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)^-1 s^-1]. ³¹P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by ³¹P NMR as a function of the external pH during growth. When the external pH was 6·8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7·4. After the external pH had decreased to 5·5, the internal pH of strain CNRZ 141 had declined by 0·6 unit, whereas that of strain CNRZ 125 had decreased by only 0·2 unit of pH.     

Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.     

Phenotypically Vif- human immunodeficiency virus type 1 is produced by chronically infected restrictive cells.

The permissivity of CD4+ transformed T cells for the replication of human immunodeficiency virus type 1 (HIV-1) vif mutants varies widely between different cell lines. Mutant vif-negative viruses propagate normally in permissive CD4+ cell lines but are unable to establish a productive infection in restrictive cell lines such as H9. As a consequence, elucidation of the function of Vif has been considerably hampered by the inherent difficulty in obtaining a stable source of authentically replication-defective vif-negative viral particles produced by restrictive cells. vif-negative, vpr-negative HIV-1 strain NDK stock, produced by the permissive SupT1 cell line, was used to infect restrictive H9 cells. By using a high multiplicity, infection of H9 cells was achieved, leading to persistent production of viral particles displaying a dramatically reduced infectious virus titer when measured in a single-cycle infectivity assay. Although these viral particles were unable to further propagate in H9 cells, they could replicate normally in CEM and SupT1 cells. Comparison of unprocessed and processed Gag proteins in the persistently produced vif-negative viral particles revealed no defect in the processing of polypeptide precursors, with no inversion of the Pr55gag/p24 ratio. In addition, there was no defect in Env incorporation for the vif-negative viral particles. Despite their apparently normal protein content, these particles were morphologically abnormal when examined by transmission electron microscopy, displaying a previously described abnormally condensed nucleoid. Chronically infected restrictive cell lines producing stable levels of phenotypically vif-negative HIV-1 particles could prove particularly useful in further studies on the function of Vif in the virus life cycle.     

Two modules from the hypersuppressive rho mitochondrial DNA are required for plasmid replication in yeast

In the yeast hypersuppressive (HS) rho⁻ mutants most of the mitochondrial genome is deleted, but the remainder containing one of the three rep sequences is amplified. One of these sequences, rep2, and its flanking regions have been previously cloned and reported to promote autonomous plasmid replication in yeast. The present study suggests that the Ars activity associated with this HS rho⁻ mitochondrial DNA (mtDNA) fragment is due to the presence in cis of at least two modules: (i) the 11-bp consensus sequence 5′-^A_TAAA^C_TATAAA^A_T-3′, common to several ars sequences, and (ii) a palindromic sequence of the mitochondrial replicator. Proper spacing between the two modules, which varies from about 100 to 200 bp, is required for the Ars ⁺ activity.     

Secondary trisomy 1 q due to a reciprocal maternal translocation]

The authors report a case of partial trisomy 1 q due to a maternal balanced translocation : t(1 ; 4) (q 32 : p 16). The evocative malformations of trisomy 1 q and monosomy 4 p are discussed and compared to seven others from the literature. Then the interest of the chromosomical prenatal diagnosis and the significance of familial genetic studies are showed.