Starch mobilization in leaves

Starch mobilization is well understood in cereal endosperms, but both the pathway and the regulation of the process are poorly characterized in other types of plant organs. Arabidopsis leaves offer the opportunity for rapid progress in this area, because of the genomic resources available in this species and the ease with which starch synthesis and degradation can be monitored and manipulated. Progress in understanding three aspects of starch degradation is described: the role of disproportionating enzyme, the importance of phosphorolytic degradation, and new evidence about the involvement of a starch-phosphorylating enzyme in the degradative process. Major areas requiring further research are outlined.     

Direct phenotypic analysis of human MHC class I antigen presentation: visualization,quantitation, and in situ detection of human viral epitopes using peptide-specific,MHC-restricted human recombinant antibodies

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8(+) T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.     

Estimating hypermutation rates from clonal tree data

To understand the mechanisms underlying the varying patterns of mutations that occur during immune and autoimmune responses, estimates of the somatic hypermutation rate are critical. However, despite its significance, precise estimates of the mutation rate do not currently exist. Microdissection studies of mutating B cell clones provide an opportunity to measure this rate more accurately than previously possible. Each microdissection provides a number of clonally related sequences that, through the analysis of shared mutations, can be genealogically related to each other. The shape of these clonal trees is influenced by many processes, including the hypermutation rate. We have developed two different methods to estimate the mutation rate based on these data. These methods are applied to two sets of experimental data, one from an autoimmune response and one from the antihapten response to (4-hydroxy-3-nitrophenyl)acetyl (NP). Comparable mutation rates are estimated for both responses, 0.7-0.9 x 10(-3) and 0.9-1.1 x 10(-3) bp(-1) division(-1) for the autoimmune and NP responses, respectively. In addition to comparing the results of the two procedures, we investigate the effect on our estimate of assumptions, such as the fraction of lethal mutations.     

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck,and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.     

An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8(+) T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen's developmental cycle. In addition to their use as candidate Ags for stimulating CD8(+) T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8(+) T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8(+) T cells specific for an H-2D(b)-restricted epitope from CrpA are elicited at a significant level (approximately 4% of splenic CD8(+) T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.     

The myelin-axolemmal complex: biochemical dissection and the role of galactosphingolipids

Myelin-axolemmal interactions regulate many cellular and molecular events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochemical fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non-compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin-axolemmal complex, a high-affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid-deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins observed in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.