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A new process to form fibroin spongy porous 3-D structure is reported herein. The process involves freezing and thawing fibroin aqueous solution in the presence of a small amount of an organic solvent. The process requires no freeze-drying, chemical cross-linking, or the aid of other polymeric materials. The solvent concentration, fibroin concentration, freezing temperature, and freezing duration affect the sponge formation, its porous structure, and its mechanical properties. Measurements by XRD and FTIR indicate that silk I and silk II crystalline structures exist in the fibroin sponge and that the secondary structure of fibroin is transformed to a β-sheet from a random coil during this process. The tensile strength decreased slightly, but the fibroin sponge showed no deformation after autoclaving. Therefore, the fibroin sponge was sterilized using an autoclave. For 3 weeks, MC3T3 cells proliferated in the sterilized fibroin sponge. The fibroin sponge formed by this new process is applicable as a tissue-engineering scaffold because it is formed from biocompatible pure silk fibroin and offers both porous structure and mechanical properties that are suitable for cell growth and handling.

We present a statistical method for estimating gene networks and detecting promoter elements simultaneously. When estimating a network from gene expression data alone, a common problem is that the number of microarrays is limited compared to the number of variables in the network model, making accurate estimation a difficult task. Our method overcomes this problem by integrating the microarray gene expression data and the DNA sequence information into a Bayesian network model. The basic idea of our method is that, if a parent gene is a transcription factor, its children may share a consensus motif in their promoter regions of the DNA sequences. Our method detects consensus motifs based on the structure of the estimated network, then re-estimates the network using the result of the motif detection. We continue this iteration until the network becomes stable. To show the effectiveness of our method, we conducted Monte Carlo simulations and applied our method to Saccharomyces cerevisiae data as a real application.
Contact: tamada@ims.u-tokyo.ac.jp

The near-surface structure and the wettability of silk fibroin films cast from aqueous solutions on hydrophobic polystyrene substrates at various temperatures is investigated by Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) and measurement of contact angle. The FTIR data reveal that the near-surface region of the films is enriched in random coil conformations of the protein at the expense of a reduced fraction of α-helix and β-sheet conformations. The relative random coil/β-sheet content shows a marked dependence on the casting temperature, displaying a minimum at 50 °C. The minimum occurs concurrently with a maximum in the wettability of film surfaces by polar liquids. In the lower wettability region, the film surfaces of this hydrophilic protein are hydrophobic, whereas in the enhanced wettability range they are slightly hydrophilic. The experimental data indicate that during formation of fibron films, α-helix and β-sheet structures are rejected by the interface because of their non-surface-active character, whereas random coils are energetically favored because at the interface they convert into a surface-active conformation which effectively minimizes the interfacial free energy and renders the polymer surface hydrophobic. In the narrow range of casting temperatures centered at 50 °C, the effect of the interface is overweighed by the bulk thermodynamics favoring the β-sheet crystallization of fibroin. Though the interfacial conformation is not accessible by FTIR-ATR, its surface-active character in combination with the unique composition and amino acid sequence of fibroin allows one to conclude that the possible chain structure is one that separates the hydrophobic alanine and hydrophilic serine residues to opposite sides of the plane passing through the chain axis.

Interferon-alpha (IFN-alpha) suppresses hepatitis B virus (HBV) gene expression by reducing its enhancer-1 activity. IFN-alpha induces transcription factors, interferon-stimulated gene factor 3 (ISGF3), and interferon regulatory factor-1 (IRF-1), which activate interferon-inducible gene expression through binding to the interferon-stimulated regulatory element (ISRE) "AGTTTCNNTTTCNC" in the gene promoters. We found the ISRE-like sequence "AGGCTTTCACTTTCTC" in the HBV enhancer-1 region and elucidated the role of this sequence. Gel mobility shift assay showed binding of in vitro translated IRF-1 and in vitro translated p48 (ISGF3-gamma), which is a component of ISGF3 to this sequence. However, nuclear extracts binding to this sequence from human hepatoma cells (HuH-7) treated with IFN-alpha contained only the protein consisted of p48. In transfection experiments, IFN-alpha suppressed the HBV enhancer-1 activity, and overexpression of p48 enhanced this inhibitory effect. Both mutation and deletion of the ISRE-like sequence in the HBV enhancer-1 region reduced the suppressive effect of IFN-alpha. Our results suggest that the ISRE-like sequence in the HBV enhancer-1 can interact with the protein containing p48 and mediate the IFN-alpha-induced suppression of the enhancer activity.

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry andin situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence.