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D Strehlow

Publications and source records attributed to D Strehlow.

11 recordsLinked to original sources
PubMed2001

Apoptosis-inducing membrane vesicles. A novel agent with unique properties.

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.

3T3 Cells↗
PubMed2001

First steps in the phytochrome phototransformation: a comparative femtosecond study on the forward (Pr --> Pfr) and back reaction (Pfr --> Pr).

The primary light-induced events in the reversible Pr right harpoon over left harpoon Pfr phototransformation are investigated by femtosecond absorption spectroscopy using a pump-probe technique. After the selective electronic excitation of Pr and Pfr with pulses at 610 and 730 nm, respectively, the transient absorption spectra were measured as a function of the delay time and subjected to a global fit analysis. As a result of this analysis, the decay-associated spectra of the kinetic components involved in the formation of the first photoproducts in the forward and back reaction are obtained. These spectra provide a more detailed understanding of the primary stages in the light-induced transformations. In addition, the influence of the solvent viscosity on the initial reaction steps was studied. In each direction of reaction, a short-lifetime component is found to be strongly viscosity-dependent, indicating that the primary photochemistry encompasses intramolecular motions of the chromophore or its proximal amino acid side chains. H-D exchange has no significant effect on the kinetics of the initial photoprocesses. This suggests that the isomerization reaction in both directions is not accompanied by a rate-limiting proton transfer.

Avena↗
PubMed2000

CD95 (Fas) ligand-expressing vesicles display antibody-mediated, FcR-dependent enhancement of cytotoxicity.

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.

Adjuvants, Immunologic↗
PubMed2000

Bioactivities of Fas ligand-expressing retroviral particles.

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction >/=500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the >/=500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l -lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.

3T3 Cells↗
PubMed2000

Retroviral membrane display of apoptotic effector molecules.

Retroviruses have been widely used in gene transmission studies. In this paper, we show that nonviral apoptotic proteins can be displayed on viral membrane surfaces and that the displayed proteins can execute their normal effector functions. We introduced the genes encoding the apoptosis effector proteins, human CD95 ligand (hFasL) or human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL), into a cell line that packages Moloney murine leukemia virus vectors. Retrovirus preparations from these lines killed target cells efficiently, and target killing was prevented by Fas-Ig fusion protein or soluble TRAIL receptor (sDR5), respectively. We show that the virus preparation exhibiting Fas-specific cytotoxicity has the same density as a retrovirus, contains full-length FasL protein, and can be depleted of infectivity by immunoadsorption with anti-FasL antibody. This novel property of retroviruses-the display of functional effector proteins-may allow the custom design of reagents whose normal function requires their being embedded in a membrane.

Animals↗
PubMed2000

Software for quantitation and visualization of expression array data.

Software is described that facilitates the analysis of phosphoimages from large array hybridizations. The Macintosh PowerPC-compatible application and its manual are available at no charge from http:¿people.bu.edu/strehlow. The software is compatible with both custom formats and array filters from three commercial manufacturers. It allows the rapid quantitation of every spot on images of hybridizations to large arrays. The user drags grids of squares over the spots on the image to define the coordinates of each spot, then aligns and edits the position of the grid. The software then corrects the positions as necessary and quantitates up to 27,000 spots per image. It stores the numerical values for each signal in a format called the fingerprint file. Fingerprint files can be directly averaged or compared, allowing the user to find mean values or differences in data from independent hybridization experiments. Data can be recalled from the fingerprint file and can be output in a variety of spreadsheet formats with several options for background correction. Finally, the software offers an output format that allows the convenient visualization of data points using animated, three-dimensional graphs.

Base Sequence↗
PubMed1999

A potential role for protease nexin 1 overexpression in the pathogenesis of scleroderma.

Scleroderma currently affects approximately 75,000-100,000 individuals in the United States. Fibroblasts isolated from lesional skin of scleroderma patients overexpress collagens and other matrix components, and this abnormality is maintained for multiple passages in culture. To understand the molecular basis for matrix gene overexpression, we performed a differential display comparison of fibroblasts from clinically lesional and nonlesional scleroderma skin. The results suggested that protease nexin 1 (PN1), a protease inhibitor, is overexpressed in scleroderma fibroblasts. Northern blot verification showed that lesional and nonlesional scleroderma fibroblasts had three- to five-fold increased levels of PN1 mRNA compared with healthy fibroblasts. Western analysis showed that scleroderma fibroblasts also secreted more PN1. In situ hybridization of skin biopsy specimens demonstrated PN1 expression in the dermis of four out of six scleroderma patients but no PN1 expression in the dermis of six healthy volunteers. Transient or stable overexpression of PN1 in mouse 3T3 fibroblasts increased collagen promoter activity or endogenous collagen transcript levels, respectively. PN1 mutagenized at its active site and antisense PN1 both failed to increase collagen promoter activity. These results suggest that overexpression of enzymatically active PN1 may play a pathogenic role in the development of the scleroderma phenotype.

3T3 Cells↗
PubMed1999

Fibroblast heterogeneity in physiological conditions and fibrotic disease.

Inflammation and tissue injury are strong stimuli for fibroblast activation and initiation of reparative processes. In certain disease states, pathological fibrosis occurs. Fibroblasts isolated from these diseased tissues often display a persistently abnormal phenotype characterized by increased synthesis of matrix components such as collagen. This metabolic abnormality is apparently independent of continued exposure to any pathological stimulus that may have initiated the process. Since fibroblasts are heterogeneous in proliferative capacity, in synthesis of collagen and other matrix proteins and in response to immune mediators and growth factors, clonal selection, i.e. selective increase in fibroblast subpopulations, may explain the long-term effects of acute in vivo activation on fibroblast behavior. Studies of SSc fibroblasts are consistent with clonal selection and/or clonal activation, processes that may play an important role in fibrosis in this and other disorders.

Animals↗
PubMed1998

Biology of the scleroderma fibroblast.

Abnormalities of matrix biosynthesis by systemic sclerosis fibroblasts underlie the cutaneous and visceral fibrosis seen in this disease. The fundamental basis of the abnormal fibroblast phenotype has not been determined but primary metabolic abnormalities, responses to abnormal environmental signals, and clonal selection have all been hypothesized to play a role. In the past year, evidence supporting each of these explanations was forthcoming. In a study of Choctaw Indians with a high incidence of scleroderma, it was shown that genetic linkage to fibrillin may play a role. This should be considered in light of demonstrated abnormalities of fibrillin structure in the tight skin mouse. Other studies have shown that abnormalities of transforming growth factor beta receptor levels, abnormalities of production and response to tissue inhibitor of matrix metal-loproteinase, and abnormalities of response to endothelial cell factors are all present in scleroderma fibroblasts. In addition to these studies in scleroderma fibroblasts, work elucidating the role of transcription factors Sp1, Sp3, and cKrox in regulating collagen metabolism provided important data upon which future studies may build.

Animals↗
PubMed1994

The fates of the blastomeres of the 16-cell zebrafish embryo.

We present a fate map for the 16-cell-stage blastomeres of the zebrafish embryo Brachydanio rerio. We injected high molecular weight fluorescent dextran into cleavage-stage cells to observe the contributions of the descendants of the first 16 cells to the adult. The patterns derived from these early cells are similar, but not identical among different embryos. Furthermore, two-color injections showed that sister blastomeres at the 16-cell stage regularly contribute to different sets of adult structures. A few of the scored tissues could not be mapped in a manner consistent with the predicted axes. Other tissues were mapped to several of the 16 blastomeres. Many of the tissues map with high probability to a few 16-cell-stage blastomeres. Thus, based on 112 injections in 56 different embryos, we have constructed a fate map by assigning probabilities for the contribution of each blastomere to each of 31 tissues in the 26-hour embryo.

Animals↗