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We have found that a cyclopropylpyrroloindole antibiotic, compound CC-1065 (benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1, 6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8, 8a-tetrahydro-7-methyl-4-oxocyclopropan[c]pyrrolo[3, 2-e]indol-2(1H)-yl)carbonyl]benzo[1,2-b:4, 3-b']dipyrrol-3(2H)-yl]-carbonyl]-1,6-dihydro-4-hydroxy-5-methoxy, (7bR,8aS)), forms interstrand DNA cross-links of an apparently covalent nature in HeLa S(3) cells. This compound induced interstrand cross-links at concentrations ranging from 0.1 to 1 nM/3 hr in whole cells, but these cross-links were absent or marginally low when the drug was added to cell lysates with inactivated cellular enzymes or isolated nuclei, which suggests that metabolic activation of the drug is a necessary step for DNA cross-linking to occur. In contrast, an analog of CC-1065, Bizelesin, which forms DNA-DNA cross-links by direct alkylation, induced interstrand DNA cross-links in both whole cells and in cell lysates. Interestingly, a demethoxy analog of CC-1065, Adozelesin, did not induce DNA cross-links under the same conditions. CC-1065 was found to be extremely potent in terms of concentrations required to cross-link DNA of tumor cells, and this may be related to its remarkable cytotoxic activity.

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The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of the growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN.

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The ALY-alyl/aly mouse is a new and unique animal model of primary immunodeficiency with autosomal recessive inheritance. The ALY mouse is devoid of superficial and profound lymph nodes and Peyer's patches. Furthermore, the lymphoid follicles and marginal zones are not clearly identified in the spleen. In addition to these structural defects, in the present study, we show that some B subpopulations are defective. Firstly, the thymic B lymphocytes are very rare. Secondly, the B220hi sIghi B subpopulation in the bone marrow is not detected as a clear cluster on FACS analyses. Thirdly, the B220 slg+ cells in the bone marrow are very rare in both ALY-aly/aly and ALY-alyl+ mice. By contrast, the NK activity is normal. Taken together, the ALY mouse is an invaluable model to elucidate the immunological networks between the lymphoid structures (lymph nodes, Peyer's patches, lymphoid follicles, etc.) and functions.

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We have found a new spontaneous autosomal recessive mutation in mice that causes a systemic absence of lymph nodes and Peyer's patches. The name "alymphoplasia", with the gene symbol "aly", is proposed for this mutant. The spleen of aly/aly mice is devoid of well-defined lymphoid follicles, and the thymus does not show a clear cortical-medullary distinction. The mutant homozygotes are deficient in both humoral and cell-mediated immune functions, and are highly susceptible to infections. They have a reduced level of IgM and severely depressed levels of IgG and IgA in their sera, and do not reject allogeneic skin grafts. However, they have mature T and B cells as determined from their cell surface antigens. The results of bone marrow transplantation experiments suggest a mesenchymal disorder as a possible cause of the lack of lymph nodes and of immunodeficiency in the aly mouse. The aly mutant mouse may be a useful animal model of primary immunodeficiency, as are the nu (nude) and scid (severe combined immunodeficiency) mice.

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Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.

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Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.

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