Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction

One purpose of this study was to examine the concept of T cell immunodominance employing a neonatal tolerance model. The extent to which a single T cell recognition site can represent the total T cell response to hepatitis B core Ag (HBcAg) was examined in the B10.S and B10 murine strains. It was shown that the entire B10.S T cell response to HBcAg was focused on a single immunodominant site represented by residues 120-131. This was demonstrated by exposing B10.S neonatal mice to p120-140 or p120-131, which resulted in a state of T cell tolerance to the entire HBcAg. In contrast, p120-140 contained an immunogenic T cell site for B10 mice, p129-140, but this site was nontolerogenic. Similarly, injection of p120-140 into (B10.S X B10)F1 neonatal mice resulted in tolerization of p120-131-specific, I-As-restricted T cells, but not of p129-140-specific, I-Ab-restricted T cells. The second purpose of this study was to attempt to explain the immunologic basis of an immunogenic yet nontolerogenic T cell determinant. It was shown that the p120-131 T cell site, which is immunogenic and tolerogenic in B10.S mice, could be converted into an immunogenic/nontolerogenic T cell site by a single amino acid substitution in either residue 127 or 129. Residues 127 and 129 were previously shown to be involved in interaction with MHC class II molecules (agretopic). These results demonstrated that the relative avidity of a peptide-MHC interaction can influence T cell tolerance induction. Furthermore, the results suggest that a higher threshold of peptide-MHC avidity may be required to induce T cell tolerance as compared to the threshold of peptide-MHC avidity required to immunize T cells.     

Cervical cytology quality assurance in Washington State.

The objectives of this study were to establish a profile of cervical cytology laboratories in Washington State, identify quality assurance problems amenable to correction through education or legislation, and describe differences between large and small cytology laboratories. All 43 Washington laboratories that perform cervical cytology were surveyed by mail during 1989. Completed surveys were returned by 37 (86%) of the laboratories. Nearly half (43%) of the respondents reported processing less than 10,000 Papanicolaou smears annually. Only one-third (35%) of the respondents reported participating in relevant proficiency programs. A proportion of smaller cytology laboratories were compensating their cytotechnologists on the basis of the number of slides read and allowing Papanicolaou smears to be read outside the confines of the laboratory. The results of this study suggest that cytotechnologists in some larger Washington laboratories have been exceeding work load limits recommended by professional associations. Recent legislation includes regulations that address cervical cytology quality assurance. However, continued efforts will need to be made to encourage voluntary adoption of quality control measures not addressed by this legislation.     

Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2.

The sequence of a region of the F plasmid containing the traLEKBP genes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2. The traLEK gene products of all three plasmids were virtually identical, with the most changes occurring in TraE. The TraB genes were also nearly identical except for an 11-codon extension at the 3' end of the R100-1 traB gene. The TraP protein of R100-l differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoA mutagenesis. The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2. The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer. The transfer ability of pOX38-traP474 was shown to be affected by mutations in the rfa (lipopolysaccharide) locus and in ompA in the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with the traP analogs from R100-1 and ColB2 to give an F-like phenotype. Thus, the TraP protein appears to play a minor role in conjugation and may interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmembrane complex formed by the tra operon products.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Spatial pattern,regeneration and growth rates of Brachystegia spiciformis and Julbernardia globiflora

Past work on tree-to-tree interactions in semi-arid savannas, through the use of nearest neighbour analysis, has shown both a regular dispersion pattern and a positive correlation between the size of a tree and the distance to its nearest neighbour. From these results, the importance of competition has been inferred. In the present study, tree-to-tree interactions in a mesic savanna woodland at Marondera, Zimbabwe, dominated by relatively small-sized individuals of Brachystegia spiciformis and Julbernardia globiflora, were examined. Results show that the regular dispersion pattern is uncommon; that there are few significant positive correlations between size of individual and distance to nearest neighbour; and that growth rate is not consistently correlated with size and distance of nearest neighbour. Results of a study of regeneration relative to canopy cover suggest a possible mechanism for these findings: unlike semi-arid savannas, young plants often grow in under-canopy environments so that early spacing of young trees does not occur. Significant positive correlations between the size of a tree and the distance to its nearest neighbour were found in a stand of larger, presumably older, trees, suggesting that such a relationship only develops in mesic miombo woodland through a thinning process as the trees mature. Familial clumping, which is common in the study area, may mask spatial patterns.Abbreviations RBAI Relative basal area increment     

A study into the effects of protein binding on nucleotide conformation.

In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations.     

The vacuolar H+-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification,mitochondrial function,and iron metabolism

Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects. The gef2 mutants display a petite phenotype. These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration. This phenotype is suppressed by adding large amounts of iron to the growth medium. A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%–75% relative to wild type. Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains. The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu. We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations. The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H ⁺-ATPase proteolipid subunit. These genetic and biochemical analyses demonstrate that the vacuolar H ⁺-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.