We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM ‫006ف(‬ kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM ‫005ف(‬ kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6-11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the "portability" of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low‐melting‐point agarose microbeads or plugs, and DNA purification in situ. This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed‐field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.

PR1 is a human leukocyte antigen (HLA)-A2 restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived antigen presenting cells, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific cytotoxic T lymphocytes (PR1-CTL) and the anti-PR1/HLA-A2 antibody 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an antigen become susceptible to therapies that target cross-presented antigens and suggest PR1 as a broadly expressed tumor antigen.

The Cdc25 phosphatases play key roles in cell-cycle progression by activating cyclin-dependent kinases. The latter are absent from neurons that are terminally differentiated in adult brain. However, accumulation of mitotic phosphoepitopes, and re-expression and activation of the M phase regulator, Cdc2/cyclin B, have been described in neurons undergoing degeneration in Alzheimer's disease (AD). To explain this atypical mitotic activation in neurons we investigated the Cdc2-activating Cdc25A phosphatase in human brain. The structural hallmarks of AD neurodegeneration, neurofibrillary tangles and neuritic plaques, were prominently immunolabeled with Cdc25A antibodies. In addition numerous neurons without visible structural alterations were also intensely stained, whereas control brain was very weakly positive. After immunoprecipitation from control and AD tissue, we found that the tyrosine dephosphorylating activity of Cdc25A against exogenous Cdc2 substrate was elevated in AD. Accordingly, Cdc25A from AD tissue displayed increased immunoreactivity with the mitotic phosphoepitope-specific antibody, MPM-2, and co-localized with MPM-2 immunoreactivity in AD neurons. These data suggest that Cdc25A participates in mitotic activation during neurodegeneration. The involvement of Cdc25A in cellular transformation, modulation of the DNA damage checkpoint, and linkage of mitogenic signaling to cell cycle machinery, also implicates one of these cell-cycle pathways in AD pathogenesis.

We have measured the apparent Ca2+ sensitivities of force development in skinned cardiac trabeculae at different sarcome lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca(2+)-force relations were measured at a long (2.4-microns) and a short (1.9-microns) sarcomere length. In disease, compared with the control condition, the apparent Ca2+ sensitivity was greatly diminished at a sarcomere length of 1.9 microns but not affected at all at the long length (2.4 microns). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca2+ sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length-dependent modifications in Ca2+ sensitivity. The findings raise the possibility that diabetic Ca(2+)-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca2+ sensitivity.