Rac1 Research Articles

Inhibition of superoxide production in B lymphocytes by rac antisense oligonucleotides.

Rac1 and Rac2 gene products are small GTP-binding proteins showing 92% homology to each other. According to recent studies performed in cell-free systems, Rac1 and Rac2 proteins may be involved in the activation of NADPH-oxidase, the superoxide-generating enzymatic complex active in phagocytes. Epstein-Barr virus (EBV) transformed B lymphocytes, which express rac1 and rac2 genes, also efficiently release superoxide anions when triggered by various cell surface stimuli. To investigate the regulatory role of Rac proteins in living cells, we analyzed superoxide production in response to cross-linking of surface immunoglobulins or phorbol ester treatment in human EBV-transformed B lymphocytes pretreated with Rac sense and antisense oligonucleotides. We report here that (i) the rac protein content estimated by immunoblotting can be decreased by 60% in Rac antisense pretreated cells and (ii) a strong (50-60%), dose-dependent inhibition of superoxide production is observed in antisense pretreated cells whereas cells pretreated with sense oligonucleotide are unaffected. The data presented show, for the first time in whole cells, that superoxide production is modulated by the Rac protein content, thus demonstrating the physiological role of Rac proteins in the regulation of NADPH-oxidase.

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.

Structural characterization of a rice actin gene

We have isolated and sequenced a full-length cDNA clone containing information for the rice actin gene RAc1. Transcript terminus mapping and sequence alignment between the RAc1 cDNA clone and a previously isolated RAc1 genomic clone were used to determine the structure of the RAc1 gene. This allowed us to make the first complete structural characterization of a plant actin gene. The analysis revealed the presence of a 5'-noncoding exon, separated by an intron, from the first translated exon of the RAc1 gene. This is one of the few reported cases of a plant gene containing such a 5'-noncoding exon. Sequence comparison between the previously isolated plant actin genes suggests that such an exon may be a common feature of plant actin gene structure. The present study also confirms that the rice actin gene family is composed of at least eight unique members.

Identification of the ral and rac1 Gene Products, Low Molecular Mass GTP-binding Proteins from Human Platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.

Natural convection in vertical Bridgman configurations

Buoyancy driven convection in closed vertical cylinders heated from below and crystal growth in corresponding configurations (vertical Bridgman technique with top seeding) have been studied experimentally and theoretically. The hydrodynamic state in such configurations is described by three dimensionless numbers, the Rayleigh number Ra, the aspect ratio (height h/diameter d) and the Prandtl number Pr. Two different types of melts have been investigated, H 2O (Pr = 6.7), and Ga and GaSb melts (Pr ≈ 2 × 10 -2) for various aspect ratios (0.5 ⩽ h/ d ⩽ 5) and Rayleigh numbers up to 10 8. Both experimental results and numerical analysis for the onset of convection (critical Rayleigh number Ra c1) and the state of convective flow are presented. The values of Ra c1 for 0.5 ⩽ h/ d ⩽ 5 and the symmetries of the basic flow (axial symmetry for h/ d = 0.5 and non-axial symmetry for 1 ⩽ h/ d ⩽ 5) are in good agreement with the theoretical predictions of Charlson and Sani [1]. Te-doped GaSb crystals have been grown by using the vertical Bridgman technique with top seeding. Temperatures have been measured in the GaSb melt during growth and compared with the results of our flow investigations in Ga melts. It shows clearly, that the occurrence of doping striations in the GaSb crystals can be very well correlated to the unsteady flow regimes.

Free Convection Heat Transfer from the Outside of Radial Fin Tubes

Heat transfer rates for a variety of finned tubes in water and asphalt-water emulsion were determined experimentally. From these data, free convection heat transfer coefficients on the outside of the tube were calculated as a function of the Rayleigh number. A correlation of the form Nu = C1(Ra)C2 was then determined by a least-squares fit of the data.