Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo. The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (Leu͞Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S. typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S. typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy. red fluorescent protein ͉ bacterial therapy ͉ auxotrophy ͉ leucine ͉ arginine

Abstract. The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.

We have recently shown that the expression of nestin, the neural stem cell marker protein, is expressed in bulge-area stem cells of the hair follicle. We used transgenic mice with GFP expression driven by the nestin regulatory element [nestin-driven GFP (ND-GFP)]. The ND-GFP stem cells give rise to the outer-root sheath of the hair follicle as well as an ND-GFP interfollicular vascular network. In this study, we demonstrate that ND-GFP stem cells isolated from the hair-follicle bulge area that are negative for the keratinocyte marker keratin 15 can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. These pluripotent ND-GFP stem cells are positive for the stem cell marker CD34, as well as keratin 15-negative, suggesting their relatively undifferentiated state. The apparent primitive state of the ND-GFP stem cells is compatible with their pluripotency. Furthermore, we show that cells derived from ND-GFP stem cells can differentiate into neurons after transplantation to the subcutis of nude mice. These results suggest that hair-follicle bulge-area ND-GFP stem cells may provide an accessible, autologous source of undifferentiated multipotent stem cells for therapeutic application.bulge area ͉ GFP ͉ differentiation ͉ glial cell ͉ smooth muscle cell ͉ transgenic mice

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stem cells ͉ hair cycle ͉ neurological ͉ GFP ͉ imaging H air growth is a unique cyclic regeneration phenomenon. The hair follicle undergoes repeated cycles of periods of growth (anagen), regression (catagen), and rest (telogen) throughout the life of mammals. The progenitor or stem cells for the outer-root sheath of the hair follicle were thought to reside in the permanent upper portion of the hair follicle, the so-called bulge area (1, 2).Recently Taylor et al. (3) reported that hair follicle bulge stem cells are potentially bipotent because they can give rise to not only cells of the hair follicle but also to epidermal cells. Other experiments (1) also have provided new evidence that the upper outer-root sheath of vibrissal (whisker) follicles of adult mice contains multipotent stem cells, which can differentiate into hair follicle matrix cells, sebaceous gland basal cells, and epidermis. Recently, Toma et al. (4) reported that multipotent adult stem cells isolated from mammalian skin dermis, termed skin-derived precursors, can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells, and adipocytes. However, the exact location of these stem cells in skin is unknown, and their functions are still unclear.We report here the expression of nestin, a marker for neural progenitor cells, in the cells of the follicle bulge. Nestin was linked to GFP, allowing us to observe that the nestin-containing cells formed the major part of the hair follicle each cycle. This expression of the neural stem cell protein nestin in hair follicle stem cells suggests a possible relation. Materials and Methods Nestin-GFP Transgenic Mice. Nestin is an intermediate filament (IF) gene that is a marker for CNS progenitor cells and neuroepithelial stem cells (5). Enhanced GFP (EGFP) transgenic mice carrying EGFP under the control of the nestin second-intron enhancer are used for studying and visualizing the self-renewal and multipotency of CNS stem cells (5-7). Here we report that hair follicle stem cells strongly express nestin as evidenced by nestin-regulated EGFP expression.Induction of Anagen. Nestin-regulated GFP transgenic mice, 6 -8 weeks old, in the telogen phase of hair growth were depilated by a hot mixture of rosin and beeswax. Samples (5 ϫ 5 mm 2 ) were excised from the dorsal skin right before depilation (telogen) and at days 1-5 (early anagen), days 8 and 10 (middle anagen), days 14 and 15 (late anagen), and days 19 and 20 (catagen) after depilation. The skin samples were divided into two parts, one for f luorescence microscopy and the other for frozen sections. Brief ly, the skin samples were embedded in tissue-freezing embedding medium and frozen at Ϫ80°C overnight. Sections 8 m thick were cut with a Leica CM1850 cryostat. The frozen sections were air-dried and counterstained with propidium iodide for f luorescence microscopy.Fluorescence and Confocal Microscopy. The nestin-GFP skin samples, after dissecting out the s.c. tissue, were directly observed with dermis up and epidermis down under a N...

Abstract. The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged.Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E.G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins.Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles. This RNP-depleted nuclear matrix is markedly distorted in overall shape when compared to the RNP-containing nuclear matrix.C OMPARED to the detailed knowledge of the arrangements of defined sequences within DNA, little is known of its orga'nization in the nucleus. The interior of the eukaryotic nucleus and its composition has been particularly difficult to study. There is strong evidence of a nuclear skeleton or matrix, but its nature a...

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization . We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis . First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer . The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely 'unaltered by detergent extraction . Residual desmosomes, as can be seen in thin sections, were also well-preserved . The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation . The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders . This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts . Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold . Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders . The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions . The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold .A potentially powerful addition to the study of cell structure is afforded by combining detergent extraction with unembedded whole mount electron microscopy. In this protocol, soluble proteins are extracted with non-ionic detergent under near physiological conditions of ionic strength and pH. The cellular structure that remains after detergent extraction is called the skeletal framework (1, 2). The elaborate filament framework can be clearly seen using whole mount transmission electron microscopy, which omits conventional embedding, sectioning, and staining. We have observed the three-

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