Growth of tissue-engineered human nasoseptal cartilage in simulated microgravity.

OBJECTIVE: To evaluate the feasibility of in vitro fabrication of tissue-engineered cartilage from human nasoseptal chondrocytes for autologous reconstruction. DESIGN: Hyaline cartilage was reconstituted from chondrocyte-polyglycolic acid scaffolding constructs in a 3-dimensional mammalian cell culture cascade. This included monolayer cellular amplification, cell seeding in the spinner flask, and tissue growth in simulated microgravity. RESULTS: The quality of the fabricated cartilage analogue was found to depend on the initial cell density, duration of incubation, and bioreactor type. Dynamic seeding was nearly completed within the first 10 hours of inoculation regardless of the cell source (cryogenically preserved vs fresh chondrocytes) or presence of serum. A duration of incubation in excess of 4 weeks was required for complete matrix biosynthesis at low seeding densities in the spinner flasks. Seeding densities greater than 2.3x10(6) chondrocytes per scaffold were required for early hyaline cartilage formation as well as longer-time mature matrix regeneration. In addition, maintaining the structural integrity of the unreinforced scaffold, which is necessary for continued mature matrix regeneration, was achievable through postseeding tissue growth in simulated microgravity. CONCLUSION: Once combined with polyglycolic acid scaffolds in the bioreactor cascades that allow efficient seeding and quiescent tissue growth, human septal chondrocytes become a valuable source of reproducible ex vivo cartilage regeneration in the laboratory.

An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction.

SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

Molecular basis of mammalian sexual determination: activation of Müllerian inhibiting substance gene expression by SRY.

The pathway of male sexual development in mammals is initiated by SRY, a gene on the short arm of the Y chromosome. Its expression in the differentiating gonadal ridge directs testicular morphogenesis, characterized by elaboration of Müllerian inhibiting substance (MIS) and testosterone. SRY and MIS each belong to conserved gene families that function in the control of growth and differentiation. Structural and biochemical studies of the DNA binding domain of SRY (the HMG box) revealed a protein-DNA interaction consisting of partial side chain intercalation into a widened minor groove. Functional studies of SRY in a cell line from embryonic gonadal ridge demonstrated activation of a gene-regulatory pathway leading to expression of MIS. SRY molecules containing mutations associated with human sex reversal have altered structural interactions with DNA and failed to induce transcription of MIS.

The C-terminus of the homeodomain is required for functional specificity of the Drosophila rough gene.

In contrast to most Drosophila homeobox genes, which are required during embryogenesis, the rough gene is involved in photoreceptor cell specification in the compound eye. Taking advantage of the viability of null rough alleles and the small size of the rough gene, we have combined in vivo and in vitro mutagenesis to define important functional domains in the rough protein. All missense mutations found to disrupt rough function mapped to highly conserved amino acids in the homeodomain (HD), suggesting that the nature of few, if any, single amino acids outside the HD is critical for rough activity. The analysis of chimeric proteins, in which the whole HD or parts of it were swapped between the rough and Antennapedia (Antp) proteins, revealed that the C-terminus of the rough HD is important for rough activity in vivo. This C-terminal region was also found to be required for the recognition of rough binding sites in vitro. Our data suggest that amino acids located in the C-terminus of the homeodomain may play important roles in selective binding site recognition.

Molecular dynamics simulations of B-DNA: an analysis of the role of initial molecular configuration, randomly assigned velocity distribution, long integration times, and nonconstrained termini.

Molecular dynamics simulations of three DNA sequences using the AMBER 3.0 force field were performed with implicit inclusion of water through a distance-dependent dielectric constant and solvated counterions. Simulations of the self-complementary DNA dodecamer d(CGCGAATTCGCG) were started from a regular B-DNA structure and the x-ray single crystal B-DNA structure. Although mean convergence during the 89-ps calculation was confirmed, localized differences in backbone torsionals and base-pair helicoidals were observed. A nanosecond simulation of the nonself-complementary 14 base-pair DNA d(GGCGGAATTGGCGG) indicates that most structural parameters stabilize within the first 100-200 ps, while isolated features show low-frequency oscillations throughout the calculation. The lack of harmonic constraints on the ends of the molecules was shown not to perturb the structural dynamics of the internal oligonucleotide beyond the external 2 base pairs. Comparison of three simulations of the nonself-complementary 14 base-pair DNA d(GGCGAAATTCGCGG), identical in all respects other than the assignment of initial Maxwellian atomic velocity distributions, revealed the inherent systematic variability. The three calculations result in nearly superimposable global structures, with localized variations in torsionals and helicoidals. Our results provide a basis for performing a comparative analysis of the effect of DNA sequence on localized structure.