Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.     

Quantifying efficacy of chemotherapy of brain tumors with homogeneous and heterogeneous drug delivery

Gliomas are diffuse and invasive brain tumors with the nefarious ability to evade even seemingly draconian treatment measures. Here we introduce a simple mathematical model for drug delivery of chemotherapeutic agents to treat such a tumor. The model predicts that heterogeneity in drug delivery related to variability in vascular density throughout the brain results in an apparent tumor reduction based on imaging studies despite continual spread beyond the resolution of the imaging modality. We discuss a clinical example for which the model-predicted scenario is relevant. The analysis and results suggest an explanation for the clinical problem of the long-standing confounding observation of shrinkage of the lesion in certain areas of the brain with continued growth in other areas.     

Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes

The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H(2)-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.     

Synthesis and first biological evaluation of 1-aza-9-oxafluorenes as novel lead structures for the development of small-sized cytostatics

A first series of novel 1-aza-9-oxafluorenes has been prepared from 3-carbonyl substituted 1,4-dihydropyridines and p-benzoquinone as small-sized cytostatics. Biological evaluation has been carried out in various cancer cell-lines. First structure-activity relationships proved the 4-phenyl substituent to be more favorable than the 4-methyl substituent. Cytostatic properties are discussed.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.     

Genetic evidence for the phylogenetic relationship between Na-Dene and Yeniseian speakers

Ruhlen's hypothesis, based on linguistic evidence, for a common phylogenetic origin of Na-Dene and Yeniseian speakers is tested using genetic data. Gene frequency data for the Kets, the only surviving Yeniseian speakers, were collected during a field study in 1993. Data for several Na-Dene groups, as well as other New World and Siberian populations, were compiled from the literature. These data were analyzed using R-matrix, principal components analysis, and Mantel tests. In a comparison of 10 New World and Siberian populations using eight alleles, 55.8% of the variation was accounted for by the first principal component, and 22.1% of the variation was subsumed by the second principal component. Contrary to Ruhlen's interpretation of the linguistic data, analysis of the genetic data shows that the Na-Dene cluster with other Native American populations, while the Kets genetically resemble the surrounding Siberian groups. This conclusion is further supported by correlations that are higher when the Kets are considered unrelated to Na-Dene speakers, and an insignificant partial correlation between genes and language when geography is held constant, indicating that spatial patterning accounts for most of the variation present in these populations.     

A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter

Using a previously described human keratin 14 (K14) promoter, we created mice expressing a peptide Ag (OVAp) in epithelial cells of the skin, tongue, esophagus, and thymus. Double transgenic mice that also express a TCR specific for this Ag (OT-I) showed evidence for Ag-driven receptor editing in the thymus. Surprisingly, such mice exhibited a severe autoimmune disease. In this work we describe the features of this disease and demonstrate that it is dependent on CD8 T cells. Consistent with the Ag expression pattern dictated by the human K14 promoter, an inflammatory infiltrate was observed in skin and esophagus and around bile ducts of the liver. We also observed a high level of TNF-alpha in the serum. Given that Ag expression in the thymus induced development of T cells with dual TCR reactivity, and that dual-reactive cells have been suggested to have autoimmune potential, we tested whether they were a causal factor in the disease observed here. We found that OT-I/K14-OVAp animals on a recombinase-activating gene-deficient background still suffered from disease. In addition, OT-I animals expressing OVA broadly in all tissues under a different promoter did not experience disease, despite having a similar number of dual-specific T cells. Thus, in this model it would appear that dual-reactive T cells do not underlie autoimmune pathology. Finally, we extended these observations to a second transgenic system involving 2C TCR-transgenic animals expressing the SIY peptide Ag with the hK14 promoter. We discuss the potential relationship between autoimmunity and self-Ags that are expressed in stratified epithelium.