Determination of stromal signatures in breast carcinoma

Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.     

An amyloid-like C-terminal domain of thrombospondin-1 displays CD47 agonist activity requiring both VVM motifs

Two VVM-containing peptides in the C-terminal domain (CBD) of thrombospondin-1 function as CD47 agonists. A recombinant form of the CBD (rCBD) has been expressed that contains both VVM sites and exhibits CD47-dependent binding of C32 melanoma cells when coated at concentrations 100x lower than the peptide 4N1K (kRFYVVMWKk). Circular dichroism and thioflavin T binding of a recombinant form of the C-terminal domain (rCBD) of thrombospondin-1 indicated a species highly enriched in beta-sheet secondary structure, with spectra similar to those of amyloid proteins. Reduction of the CD signal with progressively higher concentrations of guanidine hydrochloride was correlated with a loss of cell-binding activity. Melanoma cell spreading on vitronectin was strongly stimulated by immobilized rCBD co-coated at concentrations more than 50x lower than 4N1K, and the effect was blocked by treatment with pertussis toxin, consistent with the known mediation of CD47 signaling by trimeric G(i). Mutations of either or both VV sequences of rCBD (1037-38 and 1123-24 of TSP1) to GG had a modest effect on cell binding, a component of which was inhibited by heparin. However, all three mutants dramatically reduced the signaling-dependent stimulation of cell spreading, indicating that the VVM motifs of rCBD are structurally linked in CD47 activation.     

rHuKGF ameliorates symptoms in DSS and CD4(+)CD45RB(Hi) T cell transfer mouse models of inflammatory bowel disease

There is an acute need for effective therapy for inflammatory bowel disease (IBD), particularly at the level of repair of the damaged epithelium. We evaluated the efficacy of recombinant human keratinocyte growth factor (rHuKGF) in both the dextran sodium sulfate (DSS) and the CD4(+)CD45RB(Hi) T cell transfer models of IBD. Disease was induced either by the ad libitum administration to normal mice of 4% DSS in the drinking water or by the injection of 4 x 10(5) CD4(+)CD45RB(Hi) T cells into immunodeficient scid/scid mice. rHuKGF was administered by subcutaneous injection at doses of 1.0 or 3.0 mg/kg in both preventative and therapeutic regimens during both studies. rHuKGF significantly improved survival and body weight loss in the DSS model in both preventative and therapeutic dosing regimens. It also improved diarrhea, hematochezia, and hematological parameters, as well as large intestine histopathology. In the T cell transfer model, rHuKGF improved body weight loss, diarrhea, and levels of serum amyloid A, as well as large intestine histopathology. In both models of IBD, the colonic levels of intestinal trefoil factor (ITF) were elevated by the disease state and further elevated by treatment with rHuKGF. These data suggest that rHuKGF may prove useful in the clinical management of IBD and its effects are likely mediated by its ability to locally increase the levels of ITF.     

Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally. Bioelectromagnetics 18:264–272, 1997. © Wiley-Liss, Inc.     

Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.     

Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio

A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.     

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell–cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell–cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β₁-integrin and EWI-F were co-clustered with CD9 at cell–cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell–cell fusion by controlling the access of the fusion machinery to cell contact areas.     

Invasive species alter ontogenetic shifts in the trophic ecology of Lake Sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) in the Niagara River and Lake Ontario

Species invasions can alter food web structure and change ecosystem-level functioning, but it is often unclear how these invasions may affect the life history of native species. The Lake Sturgeon (Acipenser fulvescens), a large long-lived native fish species in the Great Lakes, has increased in abundance in the lower Niagara River and nearby Lake Ontario during a period of invasive species-induced ecosystem change precipitated most recently by Dreissenid mussels (Driessena polymorpha and Driessena bugensis) and Round Goby (Neogobius melanostomus). Material taken from cross-sections of archived pectoral spines from Niagara River Lake Sturgeon captured in 1998–2000 and 2010–2012 were analyzed for stable isotopes across discrete growth zones to provide an ontogenetic assessment of diet, and diet analysis of Lake Sturgeon captured in 2014 was conducted to assess the contribution of invasive prey. Round Goby was the most important Lake Sturgeon prey item (86% by weight) in 2014, which corroborated results of δ¹⁵N and δ¹³C. Lake Sturgeon captured after the invasion of Round Goby exhibited ontogenetic changes in δ¹⁵N that differed from pre-Round Goby patterns, though this effect was weaker for δ¹³C. Values of δ¹⁵N from spine growth zones indicated non-linear increases in trophic position with age and increased rate of δ¹⁵N enrichment after the Round Goby invasion. We conclude that Round Goby establishment in western Lake Ontario changed the feeding ecology of Lake Sturgeon, which may have a positive effect on population growth for this native species.