Amino acid sequence of a non-specific wheat phospholipid transfer protein and its conformation as revealed by infrared and Raman spectroscopy. Role of disulfide bridges and phospholipids in the stabilization of the alpha-helix structure.

A wheat non specific phospholipid transfer protein has been isolated from wheat seeds and its amino acid sequence reveals that it is composed of 90 residues for a molecular weight of 9607. From the comparison of its sequence with those of the eight known proteins of the same family, hypotheses on the role of some conserved residues in the transfer activity can be made. The conformation of this protein has been studied by Raman and Fourier transform infrared spectroscopy and this is the first report on the structure of non specific plant phospholipid transfer proteins. As opposed to previous studies on the structure prediction from the amino acid sequence, the results obtained show that plant non specific phospholipid transfer proteins are not almost entirely composed of beta-sheets. Instead, infrared results show that the wheat protein contains 41% alpha-helix and 19% beta-sheet structures, while 40% of the conformation is undefined or composed of turns. Raman spectroscopy shows that three disulfide bridges adopt a gauche-gauche-gauche conformation while the other exhibits a gauche-gauche-trans conformation, and that the two tyrosine residues are hydrogen bonded to water molecules. The cleavage of the disulfide bonds affects significantly the conformation of the protein, the extended confirmation being increased by 15% at the expense of the alpha-helix content. On the other hand, the binding of 1-palmitoyllysophosphatidylcholine to the protein leads to an increase of 8% of the alpha-helix content compared to the free protein. Secondary structure predictions from the amino acid sequence suggest that the binding of a phospholipid stabilizes helicity of the amphipathic helices while the reduction of disulfide bonds would affect the stability of the N-terminal helix. The extended structure located at the C-terminus is not affected. Finally, the wheat phospholipid transfer protein has no effect on the thermotropic behavior of large unilamellar vesicles of dimyristoylphosphatidylcholine while it increases the conformational order of the acyl chains of large unilamellar vesicles of dimyristoylphosphatidylglycerol in the liquid-crystalline state. No major conformational changes of the protein are observed when it is adsorbed to phospholipid vesicles. These results suggest that the helical structure is essential for the transfer activity without excluding a possible role of the C-terminal extended structure on the adsorption to phospholipid vesicles.     

Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization.

Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.     

Associations of esterase 6 allozyme and activity variation with reproductive fitness inDrosophila melanogaster

Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.     

Spatial and temporal variation in carbon isotope discrimination in prairie graminoids

We present the results of a 5-year examination of variation in the stable carbon isotope composition () of three C₃ graminoid species from a Sandhills prairie: Agropyron smithii, Carex heliophila and Stipa comata. Although consistent species-specific patterns for mean were seen, there was also significant and substantial among-year and within-season variation in . A smaller contribution to variation in came from topographic variation among sampling sites. Effects of species, year, season and topography contribute to variation in in an additive manner. An association between climate and exists that is consistent with previous work suggesting that reflects the interplay between photosynthetic gas exchange and plant water relations. Within the growing season, the time over which integrates plant response to the environment ranges from days to months.     

The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex

In plant development, leaf primordia are formed on the flanks of the shoot apical meristem in a highly predictable pattern. The cells that give rise to a primordium are sequestered from the apical meristem. Maintenance of the meristem requires that these cells be replaced by the addition of new cells. Despite the central role of these activities in development, the mechanism controlling and coordinating them is poorly understood. These processes have been characterized in the Arabidopsis mutant forever young (fey). The fey mutation results in a disruption of leaf positioning and meristem maintenance. The predicted FEY protein shares significant homology to a nodulin and limited homology to various reductases. It is proposed that FEY plays a role in communication in the shoot apex through the modification of a factor regulating meristem development.     

Specificity and function of monoclonal antibodies directed against Ewing sarcoma cells

A selection of 16 monoclonal antibodies has been produced against a fresh Ewing's sarcoma (ES) tumor mixed with a permanent ES cell line. The majority of antibodies identify an 80-kDa molecule, which is not detected on healthy tissues except on certain cultured monocytes. One antibody recognize the CD2 ligand MIC2 and 2 antibodies (numbers 13 and 16) define a higher-molecular-mass antigen. Antibody 16 is also expressed on mesenchymal fibroblasts of bone marrow or fetal origin. Tumorspecific antigen expression is potentially linked to the chromosome 22 abnormality decribed in Ewing's sarcoma.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.