Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death as well as expression of proinflammatory genes such as CXCL8 in malignant human astrocytoma cells. However, the molecular mechanisms that determine the fate of cells are not yet understood. The ubiquitin (Ub)-proteasome pathway regulates a wide range of cellular functions through degradation of various regulatory proteins; given this, we hypothesized that this pathway may play a central role in TRAIL-mediated signaling. We demonstrate here that inhibition of the Ub-proteasome pathway enhanced TRAIL-mediated cell death of human astrocytoma CRT-MG cells within hours by blocking degradation of active caspase-8 and -3. Proteasome inhibitors suppressed TRAIL-mediated activation of NF-B; however, inhibition of the NF-B pathway alone was not sufficient to enhance TRAIL-mediated cell death. Collectively, these results suggest that the Ub-proteasome pathway may play an important role as an antiapoptotic surveillance system by eliminating activated caspases as well as mediating NF-B-dependent signals.Received 30 December 2003; received after revision 9 February 2004; accepted 13 February 2004     

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling

PDGF-C is a member of the platelet-derived growth factor (PDGF) family, which signals through PDGF receptor (PDGFR) alphaalpha and alphabeta dimers. Here we show that Pdgfc(-/-) mice die in the perinatal period owing to feeding and respiratory difficulties associated with a complete cleft of the secondary palate. This phenotype was less severe than that of Pdgfra(-/-) embryos. Pdgfc(-/-) Pdgfa(-/-) embryos developed a cleft face, subepidermal blistering, deficiency of renal cortex mesenchyme, spina bifida and skeletal and vascular defects. Complete loss of function of both ligands, therefore, phenocopied the loss of PDGFR-alpha function, suggesting that both PDGF-A and PDGF-C signal through PDGFR-alpha to regulate the development of craniofacial structures, the neural tube and mesodermal organs. Our results also show that PDGF-C signaling is a new pathway in palatogenesis, different from, and independent of, those previously implicated.     

VEGF links hippocampal activity with neurogenesis, learning and memory

An enriched environment is associated with hippocampal plasticity, including improved cognitive performance and increased neurogenesis. Here, we show that hippocampal expression of vascular endothelial growth factor (VEGF) is increased by both an enriched environment and performance in a spatial maze. Hippocampal gene transfer of VEGF in adult rats resulted in approximately 2 times more neurogenesis associated with improved cognition. In contrast, overexpression of placental growth factor, which signals through Flt1 but not kinase insert domain protein receptors (KDRs), had negative effects on neurogenesis and inhibited learning, although it similarly increased endothelial cell proliferation. Expression of a dominant-negative mutant KDR inhibited basal neurogenesis and impaired learning. Coexpression of mutant KDR antagonized VEGF-enhanced neurogenesis and learning without inhibiting endothelial cell proliferation. Furthermore, inhibition of VEGF expression by RNA interference completely blocked the environmental induction of neurogenesis. These data support a model in which VEGF, acting through KDR, mediates the effect of the environment on neurogenesis and cognition.     

The origin of the anomalous superconducting properties of MgB(2)

Magnesium diboride differs from ordinary metallic superconductors in several important ways, including the failure of conventional models to predict accurately its unusually high transition temperature, the effects of isotope substitution on the critical transition temperature, and its anomalous specific heat. A detailed examination of the energy associated with the formation of charge-carrying pairs, referred to as the 'superconducting energy gap', should clarify why MgB(2) is different. Some early experimental studies have indicated that MgB(2) has multiple gaps, but past theoretical studies have not explained from first principles the origin of these gaps and their effects. Here we report an ab initio calculation of the superconducting gaps in MgB(2) and their effects on measurable quantities. An important feature is that the electronic states dominated by orbitals in the boron plane couple strongly to specific phonon modes, making pair formation favourable. This explains the high transition temperature, the anomalous structure in the specific heat, and the existence of multiple gaps in this material. Our analysis suggests comparable or higher transition temperatures may result in layered materials based on B, C and N with partially filled planar orbitals.     

Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres

Drosophila Crumbs (Crb) is required for apical-basal polarity and is an apical determinant in embryonic epithelia. Here, we describe properties of Crb that control the position and integrity of the photoreceptor adherens junction and photosensitive organ, or rhabdomere. In contrast to normal photoreceptor adherens junctions and rhabdomeres, which span the depth of the retina, adherens junctions and rhabdomeres of Crb-deficient photoreceptors initially accumulate at the top of the retina and fail to maintain their integrity as they stretch to the retinal floor. We show that Crb controls localization of the adherens junction through its intracellular domain containing a putative binding site for a protein 4.1 superfamily protein (FERM). Although loss of Crb or overexpression of the FERM binding domain causes mislocalization of adherens junctions, they do not result in a significant loss of photoreceptor polarity. Mutations in CRB1, a human homologue of crb, are associated with photoreceptor degeneration in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis (LCA). The intracellular domain of CRB1 behaves similarly to its Drosophila counterpart when overexpressed in the fly eye. Our studies may provide clues for mechanisms of photoreceptor degeneration in RP12 and LCA.     

Bandgap modulation of carbon nanotubes by encapsulated metallofullerenes

Motivated by the technical and economic difficulties in further miniaturizing silicon-based transistors with the present fabrication technologies, there is a strong effort to develop alternative electronic devices, based, for example, on single molecules. Recently, carbon nanotubes have been successfully used for nanometre-sized devices such as diodes, transistors, and random access memory cells. Such nanotube devices are usually very long compared to silicon-based transistors. Here we report a method for dividing a semiconductor nanotube into multiple quantum dots with lengths of about 10nm by inserting Gd@C82 endohedral fullerenes. The spatial modulation of the nanotube electronic bandgap is observed with a low-temperature scanning tunnelling microscope. We find that a bandgap of approximately 0.5eV is narrowed down to approximately 0.1eV at sites where endohedral metallofullerenes are inserted. This change in bandgap can be explained by local elastic strain and charge transfer at metallofullerene sites. This technique for fabricating an array of quantum dots could be used for nano-electronics and nano-optoelectronics.     

High critical current density and enhanced irreversibility field in superconducting MgB2 thin films

The discovery of superconductivity at 39 K in magnesium diboride offers the possibility of a new class of low-cost, high-performance superconducting materials for magnets and electronic applications. This compound has twice the transition temperature of Nb3Sn and four times that of Nb-Ti alloy, and the vital prerequisite of strongly linked current flow has already been demonstrated. One possible drawback, however, is that the magnetic field at which superconductivity is destroyed is modest. Furthermore, the field which limits the range of practical applications-the irreversibility field H*(T)-is approximately 7 T at liquid helium temperature (4.2 K), significantly lower than about 10 T for Nb-Ti (ref. 6) and approximately 20 T for Nb3Sn (ref. 7). Here we show that MgB2 thin films that are alloyed with oxygen can exhibit a much steeper temperature dependence of H*(T) than is observed in bulk materials, yielding an H* value at 4.2 K greater than 14 T. In addition, very high critical current densities at 4.2 K are achieved: 1 MA cm-2 at 1 T and 105 A cm-2 at 10 T. These results demonstrate that MgB2 has potential for high-field superconducting applications.     

Antibacterial agents based on the cyclic D,L-alpha-peptide architecture

The rapid emergence of bacterial infections that are resistant to many drugs underscores the need for new therapeutic agents. Here we report that six- and eight-residue cyclic d,l-alpha-peptides act preferentially on Gram-positive and/or Gram-negative bacterial membranes compared to mammalian cells, increase membrane permeability, collapse transmembrane ion potentials, and cause rapid cell death. The effectiveness of this class of materials as selective antibacterial agents is highlighted by the high efficacy observed against lethal methicillin-resistant Staphylococcus aureus infections in mice. Cyclic d,l-alpha-peptides are proteolytically stable, easy to synthesize, and can be derived from a potentially vast membrane-active sequence space. The unique abiotic structure of the cyclic peptides and their quick bactericidal action may also contribute to limit temporal acquirement of drug resistant bacteria. The low molecular weight d,l-alpha-peptides offer an attractive complement to the current arsenal of naturally derived antibiotics, and hold considerable potential in combating a variety of existing and emerging infectious diseases.