Over one million patients per year undergo some type of procedure involving cartilage reconstruction. Polymer hydrogels, such as alginate, have been shown to be effective carriers for chondrocytes in subcutaneous cartilage formation. The goal of our current study was to develop a method to create complex structures (nose bridge, chin, etc.) with good dimensional tolerance to form cartilage in specific shapes. Molds of facial implants were prepared using Silastic ERTV. Suspensions of chondrocytes in 2% alginate were gelled by mixing with CaSO(4) (0.2 g/mL) and injected into the molds. Constructs of various cell concentrations (10, 25, and 50 million/mL) were implanted in the dorsal aspect of nude mice and harvested at times up to 30 weeks. Analysis of implanted constructs indicated progressive cartilage formation with time. Proteoglycan and collagen constructs increased with time to approximately 60% that of native tissue. Equilibrium modulus likewise increased with time to 15% that of normal tissue, whereas hydraulic permeability decreased to 20 times that of native tissue. Implants seeded with greater concentrations of cells increased proteoglycan content and collagen content and equilibrium and decreased permeability. Production of shaped cartilage implants by this technique presents several advantages, including good dimensional tolerance, high sample-to-sample reproducibility, and high cell viability. This system may be useful in the large-scale production of precisely shaped cartilage implants.

These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Saccharomyces cerevisiae a and a cells express the complementary cell surface glycoproteins a-agglutinin and a-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterize a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAJ). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called agal. The agal mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA] gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by agal mutants, indicating that AGA] is involved in cell surface attachment of a-agglutinin. This result suggests that AGA] encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA] transcript was expressed and induced by pheromone in both a and a cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.The a-and a-agglutinins are cell surface glycoproteins expressed by a and a cells of Saccharomyces cerevisiae, respectively (8, 31, 58). The two agglutinins interact with one another to mediate adhesion between cells of opposite mating type during the mating process. Other species of budding yeast express analogous cell surface agglutinins (4,36,38,57,61). In each yeast species, the agglutinin of one mating type is biochemically similar to the S. cerevisiae aagglutinin, and the agglutinin of the opposite mating type is similar to the S. cerevisiae a-agglutinin. Although many of the biochemical features are conserved between the agglutinins of different yeast species, the agglutinin interactions are highly species specific (4,36,56).Analyses of a-agglutinin analogs from other yeasts have used fragments released from the cell surface by limited proteolysis or reduction of disulfide bonds. Proteolytic fragments of agglutinins from Hansenula wingei (5-agglutination factor), Pichia amethionina (a-agglutinin), and Saccharomyces kluyveri (16-agglutination factor) have molecular weights of 150,000 to several million and are 80 to 95% carbohydrate, most or all of which is 0 linked (4, 36,38,61

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Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3Ј-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (⌬⌿ m ). Because ⌬⌿ m is the driving force for mitochondrial ATP synthesis, loss of ⌬⌿ m results in depletion of cellular ATP level. The loss of ⌬⌿ m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ⌬⌿ m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.Apoptosis, a form of programmed cell death (PCD), is a genetically regulated active physiological process of cell suicide that causes cell deletion without inflammation, scarring, or release of cellular contents. Mitochondria of living cells play a pivotal role in controlling life and death (Green and Reed, 1998). Mitochondria are an important cellular source for the generation of reactive oxygen species (ROS) inside the cells (Halliwell and Gutteridge, 1990). Maintenance of proper mitochondrial transmembrane potential (⌬⌿ m ) is essential for the survival of the cell because it derives the synthesis of ATP and maintains oxidative phosphorylation (Gottlieb, 2001).3,3Ј-Diindolylmethane (DIM) is a major acid condensation product of indole-3-carbinol, a natural compound found in vegetables of the genus Brassica. It has an anticarcinogenic effect and inhibits the growth of human cancer cells. DIM-

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In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

We present an attractive new system for the specific and sensitive detection of the malaria causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer sized products detectable at the single-molecule level. Combined with a droplet-microfluidics Lab-on-a-Chip platform, this design allowed for sensitive, specific and quantitative detection of all human malaria causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in non-invasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet-microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water- or food quality control or other purposes within applied or basic science.