Tissue localization of TGFα and apoptosis are inversely related in colorectal tumors

Expression of TGFalpha and the EGF receptor was studied in relation to apoptosis in human colorectal mucosa and premalignant and malignant tumors. In normal mucosa the proteins colocalized both in the proliferation compartment and at the luminal pole of the crypts in cells committed to undergo apoptosis. While staining for the EGF receptor was increased in premalignant and malignant lesions, TGFalpha was undetectable in aberrant crypt foci as well as large areas of adenomas. Incidence of apoptosis (AI) was high in these areas ranging from 8.83-24.59. Adenomas did, however, contain islands of high TGFalpha expression where AI was decreased to a range of 0.76-4.00 (decreased at P=0.0027). In carcinomas TGFalpha expression was increased above both normal and adenoma levels corresponding to the decrease in apoptosis in the malignant tumors. Tissue localization of TGFalpha and AI were still inversely related ( P=0.022), but interpatient variability was much larger than for adenomas. The data indicate that TGFalpha is the main survival factor in premalignant tumor cells of the colon, while additional factors moderate its effect in carcinomas. This suggests the possibility of targeting the EGF receptor pathway not only for treatment but also for the reversal of adenoma growth and the prevention of malignant colorectal tumors.     

Monoclonal antibodies as therapeutics in oncology

The specificity of antibodies has been harnessed to target cancer cells and the first therapeutic antibodies for use in oncology are now finding application in the clinic. Studies are currently under way to develop new and improved antibodies. Recent developments have been made in the identification of novel targets, including the use of genomic and proteomic technologies. Several methods are also being developed to enhance antibody efficacy.     

Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis,osteoarthritis, Ehlers-Danlos syndrome,muscular dystrophy,and corneal diseases

Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.     

Huntingtin fragments that aggregate go their separate ways

N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington's disease. show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions.     

Biglycan knockout mice: new models for musculoskeletal diseases

Biglycan is a Class I Small Leucine Rich Proteoglycans (SLRP) that is localized on human chromosome Xq28-ter. The conserved nature of its intron-exon structure and protein coding sequence compared to decorin (another Class I SLRP) indicates the two genes may have arisen from gene duplication. Biglycan contains two chondroitin sulfate glycosaminoglycan (GAG) chains attached near its NH₂ terminus making it different from decorin that has only one GAG chain. To determine the functions of biglycan in vivo, transgenic mice were developed that were deficient in the production of the protein (knockout). These mice acquire diminished bone mass progressively with age. Double tetracycline-calcein labeling revealed that the biglycan deficient mice are defective in their capacity to form bone. Based on this observation, we tested the hypothesis that the osteoporosis-like phenotype is due to defects in cells critical to the process of bone formation. Our data shows that biglycan deficient mice have diminished capacity to produce marrow stromal cells, the bone cell precursors, and that this deficiency increases with age. The cells also have reduced response to tranforming growth factor- (TGF-), reduced collagen synthesis and relatively more apoptosis than cells from normal littermates. In addition, calvaria cells isolated from biglycan deficient mice have reduced expression of late differentiation markers such as bone sialoprotein and osteocalcin and diminished ability to accumulate calcium judged by alizerin red staining. We propose that any one of these defects in osteogenic cells alone, or in combination, could contribute to the osteoporosis observed in the biglycan knockout mice. Other data suggests there is a functional relationship between biglycan and bone morphogenic protein-2/4 (BMP 2/4) action in controlling skeletal cell differentiation. In order to test the hypothesis that functional compensation can occur between SLRPs, we created mice deficient in biglycan and decorin. Decorin deficient mice have normal bone mass while the double biglycan/decorin knockout mice have more severe osteopenia than the single biglycan indicating redundancy in SLRP function in bone tissue. To further determine whether compensation could occur between different classes of SLRPs, mice were generated that are deficient in both biglycan (class I) and fibromodulin, a class II SLRP highly expressed in mineralizing tissue. These doubly deficient mice had an impaired gait, ectopic calcification of tendons and premature osteoarthritis. Transmission electron microscopy analysis showed that like the decorin and biglycan knockouts, they have severely disturbed collagen fibril structures. Biomechanical analysis of the affected tendons showed they were weaker compared to control animals leading to the conclusion that instability of the joints could be the primary cause of all the skeletal defects observed in the fibromodulin/biglycan knockout mice. These studies present important new animal models for musculoskeletal diseases and provide the opportunity to characterize the network of signals that control tissue integrity and function through SLRP activity. Published in 2003.     

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system

Nucleotide excision repair in Escherichia coli involves formation of the UvrB–DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB–DNA complex. Both stable and unstable UvrB–DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB–DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix–hairpin–helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)₂ domain. This (HhH)₂ domain was previously shown to be important for the 5′ incision on a substrate containing a (cis-Pt)·GG adduct, but not for 3′ incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)₂ domain can be important for 3′ and/or 5′ incision. We propose that the (HhH)₂ domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.     

Activation of substrate/product couples by medium-chain acyl-CoA dehydrogenase

Natural substrate/product binding activates medium-chain acyl-CoA dehydrogenase (MCAD) to accept electrons from its substrate by inducing a positive flavin midpoint potential shift. The energy source for this activation has never been fully elucidated. If ground-state alterations of the ligand, such as polarization, are entirely responsible for enzyme activation, the ligand potential should shift equally to that of the flavin but in the opposite direction. Ligand polarization is likely responsible for only a small portion of this activation. Here, thiophenepropionoyl- and furylpropionoyl-CoA analogs were used to directly measure the redox modulations of several ligand couples upon binding to MCAD. These measurements identified the thermodynamic contribution of ligand polarization to enzyme activation. Because the ligand potential alterations are significantly smaller than modulations in the flavin potential due to binding, other phenomena such as pK(a) changes, desolvation, and charge alterations are likely responsible for the thermodynamic modulations required for MCAD's activity.     

In vivo evidence for interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na+/H+ exchanger knockout model of congenital diarrhea

Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon-gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.     

Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism

All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.