Prior vaccination increases the epitopic breadth of the cytotoxic T-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection

Although recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.     

Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion

During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract. In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells. We reported two molecules involved in this adhesion event. One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin. SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere. The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style. For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.     

ISMB 2003 Bio-ontologies SIG and Sixth Annual Bio-ontologies Meeting Report

The Annual Bio-Ontologies meeting (http://www.cs.man.ac.uk/ stevens/meeting03/) has now been running for 6 consecutive years, as a special interest group (SIG) of the much larger ISMB conference. It met in Brisbane, Australia, this summer, the first time it was held outside North America or Europe. The bio-ontologies meeting is 1 day long and normally has around 100 attendees. This year there were many fewer, no doubt a result of the distance, global politics and SARS. The meeting consisted of a series of 30 min talks with no formal peer review or publication. Talks ranged in style from fairly formal and complete pieces of work, through works in progress, to the very informal and discursive. Each year's meeting has a theme and this year it was 'ontologies, and text processing'. There is a tendency for those submitting talks to ignore the theme completely, but this year's theme obviously struck a chord, as half the programme was about ontologies and text analysis (http://www.cs.man.ac.uk/ stevensr/meeting03/programme.html). Despite the smaller size of the meeting, the programme was particularly strong this year, meaning that the tension between allowing time for the many excellent talks, discussion and questions from the floor was particular keenly felt. A happy problem to have!     

Effect of age on susceptibility to post-traumatic infection in the elderly

Previous work has demonstrated an age-related decline in neutrophil function, including a decline in phagocytic capacity, with age in healthy individuals. This decline in function may contribute to increased susceptibility to bacterial infections in the elderly population. The present study has investigated the effects of age on susceptibility to infection and neutrophil function in elderly humans following mild trauma. Specifically, we have measured neutrophil function in 44 patients, all of whom had no significant co-morbidity, were over 65 years old (mean age 82.5 years) and had sustained a fractured neck of femur. We obtained neutrophils and examined the process of microbial engulfment by phagocytosis and the bactericidal mechanism of superoxide production. In the 5-week period after trauma, almost half of the elderly trauma patients succumbed to bacterial or fungal infection, with a predominance of chest and urinary tract infections. When examining neutrophil function, a decline in superoxide production was observed in neutrophils from the elderly trauma group at the time of hip fracture when compared with those from healthy elderly controls, and this was maintained 5 weeks after trauma. This was accompanied by an age-related reduction in phagocytic function during this period. We propose that trauma and an age-related decline in neutrophil function combine to decrease the immune response to bacteria in the elderly.     

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.     

Cell cycle programs of gene expression control morphogenetic protein localization

Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms.     

Glycoprotein degradation: do sugars hold the key?

Secretory glycoproteins that fail to fold or assemble correctly are retained in the endoplasmic reticulum and eventually degraded. Recent evidence shows that trimming of their N-linked oligosaccharide chains plays a key role in targeting glycoproteins for destruction.     

Enhancement of tumor perfusion and oxygenation by carbogen and nicotinamide during single- and multifraction irradiation

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 x 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.     

Decreased lateral aggregation of a variant recombinant fibrinogen provides insight into the polymerization mechanism

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.