Sequence and diversity of MHC <Emphasis Type="Italic">DQA</Emphasis> and<Emphasis Type="Italic"> DQB</Emphasis> genes of the owl monkey<Emphasis Type="Italic"> Aotus nancymaae</Emphasis>

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.     

Sequence and diversity of DRB genes of Aotus nancymaae, a primate model for human malaria parasites

 The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for evaluation of malaria vaccine candidates. We present here a first step in the molecular characterization of the major histocompatibility complex (MHC) class II DRB genes of Aotus nancymaae (owl monkey or night monkey) by nucleotide sequence analysis of the polymorphic exon 2 segments. In a group of 15 nonrelated animals captivated in the wild, 34 MHC DRB alleles could be identified. Six allelic lineages were detected, two of them having human counterparts, while two other lineages have not been described in any other New World monkey species studied. As in the common marmoset, the diversity of DRB alleles appears to have arisen largely by point mutations in the β-pleated sheets and by frequent exchange of fixed sequence motifs in the α-helical portion. Pairs of alleles differing only at amino acid position b86 by an exchange of valine to glycine are present in Aotus, as in humans. Essential amino acid residues contributing to MHC DR peptide binding pockets number 1 and 4 are conserved or semiconserved between HLA-DR and Aona-DRB molecules, indicating a capacity to bind similar peptide repertoires. These results support fully our using Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. Received: 10 August 1999 / Revised: 11 October 1999     

Association of the low-density lipoprotein receptor gene with obesity in Native American populations

Five low-density lipoprotein receptor gene (LDLR) restriction fragment length polymorphisms (RFLPs: TaqI, intron 4; HincII, exon 12; AvaII, exon 13; MspI and NcoI, exon 18) were investigated in 131 individuals from five Brazilian Indian tribes. All markers were polymorphic in this ethnic group. In the whole sample of Amerindians, 13 (41%) of the 32 expected haplotypes were identified, but only three were shared by all tribes. The Xavante, Suruí, Zoró, and Gavi?o tribes, who had been studied for anthropometry, were grouped according to their genotypes, and the corresponding mean values were examined. Significant associations were observed between HincII *H-, AvaII *A+, MspI *M-, and NcoI *N+ and the body mass index (BMI), triceps and subscapular skinfolds, and the arm fat index (AFI). Haplotypes were derived for these four RFLPs, and (*H-/*A+/*M-/*N+) haplotype carriers were compared with noncarriers of this haplotype with equally significant results for the three parameters (BMI, P=0.021; skinfold thickness, P<0.001; AFI, P=0.005). These results suggest that the LDLR gene has some influence over adipose tissue deposition.     

Potassium and Sodium Fluxes in the PhytopathogenicFungus Fusarium oxysporum var. lini

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb⁺ uptake in normal-K⁺ cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30°C. Differences in the K _m values and in the Arrhenius plots of Rb⁺ uptake suggest different uptake systems in K⁺-starved and in normal-K⁺ cells. In addition, the substantial inhibition of Rb⁺ uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li⁺ uptake in K⁺-starved cells exhibits a monophasic kinetics with low affinity. Li⁺ efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na⁺/cation antiporter is not involved in this process. Received: 14 March 2000 / Accepted: 5 June 2000     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.     

Influence of high biomass concentrations on alkane solubilities

Alkane solubilities were measured experimentally for high-density biomass. The resulting Henry's law constants for propane were found to decrease significantly for both dense yeast suspensions and an actual propane-degrading biofilm consortium. At the biomass densities of a typical biofilm, propane solubility was about an order of magnitude greater than that in pure water. For example, a dense biofilm had a propane Henry's law constant of 0.09+/-0.04 atm m(3) mol(-1) compared to 0.6+/-0.1 atm m(3) mol(-1) measured in pure water. The results were modeled with mixing rules and compared with octanol-water mixtures. Hydrogels (agar) and salts decreased the alkane solubility. By considering a theoretical solubility of propane in dry biomass, estimates were made of intrinsic Henry's law constants for propane in pure yeast and biomass, which were 13+/-2 and 5+/-2 atm kg biomass mol(-1) for yeast and biofilm consortium, respectively.     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Engineering the Escherichia coli outer membrane protein OmpC for metal bioadsorption

The outer membrane protein, OmpC, from Escherichia coli was used to display metal-binding poly-histidine peptides on the surface of this bacterium. SDS-PAGE analysis of outer membrane protein preparations confirmed the expression of the metal-binding epitopes inserted in position 162 of the mature OmpC protein. Display of these epitopes was confirmed by epifluorescence microscopy of cells bound to Ni²⁺-NTA-agarose beads and metal adsorption experiments. The cells harboring one or two copies of the metal binding epitope were able to adsorb 3 to 6 times more Zn²⁺ (13.8 mol g^–1 cell), Fe³⁺ (35.3 mol g^–1 cell), and Ni²⁺ (9.9 mol g^–1 cell) metallic ions than control cells expressing the wild-type OmpC.