Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

Characteristics of the haemoproteid community in an expanding white-winged dove population

The haemoproteid community of 171 eastern white-winged doves (Zenaida asiatica asiatica) from the expanding Texas population was examined using thin blood smears. During summer 1997, heart blood was taken from doves within their historical breeding range (Lower Rio Grande Valley of Texas), an intermediate region (San Antonio and surrounding area), and the new breeding periphery (north central to southeast Texas). Two species were found: Haemoproteus columbae and Haemoproteus sacharovi. Infracommunities rarely occurred in heart blood, as only 20 of 132 infected doves demonstrated gametocytes of both species. Overall prevalence of H. columbae and H. sacharovi was 77 and 15%, respectively. Prevalence of H. columbae was higher in the Lower Rio Grande Valley (LRGV) and intermediate regions than at the periphery, higher in adults than juveniles, and similar between males and females. Prevalence of H. sacharovi was lower in the LRGV than intermediate and peripheral regions, similar between juveniles and adults, and higher in females than males. Mean density of H. columbae and H. sacharovi was 15.9 +/- 2.7 and 0.3 +/- 0.1 (mean +/- SE per 3,000 erythrocytes), respectively. Overall mean abundance of H. columbae and H. sacharovi was 12.2 +/- 2.2 and 0.04 +/- 0.02, respectively. Mean abundance of H. columbae was higher in the LRGV and intermediate regions than at the periphery and was similar between host age and between host sex; H. sacharovi was similar among regions, host age, and host sex. This study emphasizes the importance of using prevalence, density, and abundance data to assess haemoproteid community structure and pattern.     

Reversal by dithiothreitol treatment of the block in murine leukemia virus maturation induced by disulfide cross-linking

We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.     

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.     

Characterization and daily variation of nitrate reductase in Gracilaria tenuistipitata (Rhodophyta)

The chemosensory protein CSP-sg4 of the desert locust Schistocerca gregaria binds reversibly N-phenyl-1-naphthylamine in fluorescent-binding assays, with a dissociation constant of 4 microM. Upon binding to the protein, the emission peaks of the fluorescent probe undergo a marked blue shift, accompanied by an order of magnitude increase of the maximum intensity. The assay has also allowed the measurement of the affinity of CSP to other aromatic and aliphatic compounds. The binding capacity of this protein is unaffected by thermal treatments up to 100 degrees C for 20 min. The ligand-binding characteristics of chemosensory proteins may help in clarifying the role of this recently discovered class of soluble proteins in chemoreception.     

BBS4 is a minor contributor to Bardet-Biedl syndrome and may also participate in triallelic inheritance

Bardet-Biedl syndrome (BBS) is an uncommon multisystemic disorder characterized primarily by retinal dystrophy, obesity, polydactyly, and renal dysfunction. BBS has been modeled historically as an autosomal recessive trait, under which premise six independent BBS loci (BBS1-BBS6) have been mapped in the human genome. However, extended mutational analyses of BBS2 and BBS6, the first two BBS genes cloned, suggest that BBS exhibits a more complex pattern of inheritance, in which three mutations at two loci simultaneously are necessary and sufficient in some families to manifest the phenotype. We evaluated the spectrum of mutations in the recently identified BBS4 gene with a combination of haplotype analysis and mutation screening on a multiethnic cohort of 177 families. Consistent with predictions from previous genetic analyses, our data suggest that mutations in BBS4 contribute to BBS in <3% of affected families. Furthermore, integrated mutational data from all three currently cloned BBS genes raise the possibility that BBS4 may participate in triallelic inheritance with BBS2 and BBS1, but not the other known loci. Establishment of the loci pairing in triallelism is likely to be important for the elucidation of the functional relationships among the different BBS proteins.     

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.