Functional GATA-3 binding sites within murine CD8 alpha upstream regulatory sequences.

Genes encoding the accessory molecules CD8 and CD4 are activated early in thymocyte development, generating CD4+8+ double positive intermediates, which give rise to two functionally distinct mature T cell subsets that express either CD4 or CD8. The mechanisms that govern the activation or suppression of the CD8 gene are likely to be central to the T cell development program. To identify the key regulatory factors, we have initiated an analysis of the transcriptional regulation of the murine CD8 alpha gene. We have identified three CD8+ cell-specific DNAase I hypersensitive sites (HSS) located upstream of the murine CD8 alpha gene. In vitro mobility shift analysis of the -4.0-kb HSS region has revealed multiple binding sites for the T cell-restricted transcription factor GATA-3. In vitro translated murine GATA-3 binds specifically to both CD8 GATA sites, and coexpression of this factor in transient transfection assays transactivates a reporter construct containing these sequences. These results provide the first evidence for the role of a T cell-restricted factor in the regulation of either CD8 or CD4 genes.

Strain-induced effects in colloidal quantum dots: lifetime measurements and blinking statistics.

A series of samples of CdSe/Cd(x)Zn(1-x)S core/shell quantum dots have been synthesized in order to measure the influence of lattice-mismatch-induced strain on the photoluminescence (PL) and blinking behaviour. The PL spectra show a significant variation of the fluorescence wavelength even though the colloidal quantum dots (cQDs) are similar in size. The PL excitation spectra show a gradual splitting of the first exciton level as the proportion of Zn is increased in the shell and as the shell grows. On the other hand, blinking studies clearly demonstrate a significant dependence on the amount of Zn present in the shell. Distributions of on and off times go from the usual power-law distributions to power-law distributions with exponential cut-offs. These cut-offs become increasingly pronounced as the proportion of Zn increases. We interpret these results in the framework of diffusion-controlled electron transfer. Exciton relaxation lifetime measurements strongly suggest that lattice mismatch is responsible for a greater number of defects in core/shell cQDs. Therefore, strain and lattice mismatch are shown to be parameters of significant importance for the electronic structure of nanocrystals, influencing the photoluminescence, exciton relaxation lifetime and blinking behaviour.

Human Merkel cell regeneration in skin derived from cultured keratinocyte grafts.

Human Merkel cell regeneration in epidermis derived from cultured keratinocyte autografts was studied from 6 days to 6 years after transplantation. Cultured keratinocyte sheets derived from skin of the sole, axilla, groin, or scalp were transplanted to full-thickness wounds in 20 pediatric patients treated for massive burns or giant congenital nevi. Normal age- and site-matched skin as well as meshed split-thickness autografts from the same patients served as controls. Merkel cells were identified by immunohistochemistry using antibodies to cytokeratins #8 and #18. Cultured keratinocytes in vitro expressed no neuroendocrine markers, but nonspecific, simple-epithelial cytokeratin expression was observed in about 20% of cells. After transplantation, Merkel cells were identified only in cultured grafts derived from sole skin and appeared in the epidermis as early as 21 days postgrafting. Dermal Merkel cells were rarely observed, but their appearance invariably succeeded that of intraepidermal Merkel cells. Regenerated Merkel cells were never innervated, and their emergence was unrelated either spatially or temporally to epidermal reinnervation. In skin bridges of meshed split-thickness grafts, Merkel cells survived after degeneration of associated neurites, but no Merkel cells appeared within re-epithelialized interstices. Among the neuroendocrine markers tested, Merkel cells in cultured grafts, meshed skin grafts or normal pediatric skin expressed only neuron-specific enolase. They failed to stain for calcitonin, chromogranin A, Leu-7, synaptophysin, bombesin, or vasoactive intestinal polypeptide by immunohistochemistry. These findings suggest that: (a) Merkel cells derive from keratinocyte precursors which undergo neuroendocrine differentiation in the epidermis; (b) that keratinocyte stem cells are capable of undergoing Merkel cell differentiation postnatally; (c) that postnatal Merkel cell differentiation may be body-site dependent; and (d) that Merkel cell development and maintenance is independent of neural induction.

Activation of the NF-kappa B and I kappa B system in smooth muscle cells after rat arterial injury. Induction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1.

The NF-kappa B transcription factor family and its inhibitory proteins (I kappa B) form an autoregulatory system that has been linked to endothelial gene expression and vascular disease. To determine the role of the NF-kappa B/I kappa B system in smooth muscle cells (SMCs) in vivo, the present study used the balloon catheter injury model in the rat carotid artery. The NF-kappa B family members p50, p65, p52, c-Rel, and RelB as well as the inhibitor proteins I kappa B alpha, I kappa B beta, and p105 were present in uninjured arteries as determined by immunoblotting. Using electromobility shift assays, low levels of constitutively activated p50, p65, and c-Rel were seen in normal carotid arteries and a fivefold induction occurred during times of rapid SMC proliferation and neointima formation after balloon denudation. Furthermore, immediately after injury, the levels of I kappa B alpha, I kappa B beta, and p105 were dramatically reduced. Expression of the NF-kappa B-regulated genes, vascular cell adhesion molecule (VCAM)-1 and monocyte chemotactic protein (MCP)-1, was apparent in SMCs within 4 hours after injury. Macrophage infiltration occurred in parallel with the expression of VCAM-1 and MCP-1, and these inflammatory cells were present on the luminal surface of injured vessels during intimal lesion formation. In chronically denuded vessels, the SMCs on the luminal surface continued to express high levels of VCAM-1 and MCP-1, which may account for the increased presence of macrophages. Together, these findings link the activation of NF-kappa B to intimal lesion formation and to the inflammatory response associated with SMCs after vascular injury.

Cultured human sole-derived keratinocyte grafts re-express site-specific differentiation after transplantation.

Cultured epithelial autografts (CEA) derived from sole skin were transplanted to full-thickness wounds excised to muscle fascia over a variety of diverse body sites in 12 pediatric patients treated for acute burns or giant congenital nevi. The skin regenerated from the grafts was biopsied from 7 days to 6 years after grafting. The resultant epidermal phenotype was analyzed histologically and by immunohistochemical localization of keratin 9 (K9) as objective evidence of sole-type site-specific differentiation. Expression of K9 was also verified by one-dimensional gel electrophoresis of epidermal cytoskeletal extracts and K9 immunoblot analysis. Grafts prepared from epidermis of axilla; groin or foreskin and transplanted to wounds of comparable depth in an identical manner in the same patients served as controls of postgrafting differentiation. Biopsies of sole skin from amputation specimens from patients of comparable age served as normal positive controls, and biopsies of nonsole skin from patients of comparable age served as normal negative controls. As early as 2 weeks postgrafting, the histologic appearance of sole-derived CEA differed substantively from that of axilla- or groin-derived CEA controls and displayed a phenotype characteristic of sole skin with a thick compact stratum corneum, a thick stratum granulosum, and a distinct stratum lucidum. In sole-derived grafts rete ridges regenerated within 2 months postgrafting, whereas nonsole-derived grafts required 4-6 months for rete ridge regeneration. Once acquired, the sole skin phenotype was maintained long-term by all sole-derived CEA. In vitro, sole-derived keratinocytes synthesized little, if any, K9. However, within 7 days after grafting, K9 synthesis by multiple suprabasal keratinocytes was seen within the epidermis regenerated from sole-derived CEA. Protein of K9 appeared progressively more diffuse throughout the suprabasal layers, attaining a confluent pattern of expression comparable to normal controls of sole skin by 6 to 12 months postgrafting, and the confluent pattern of suprabasal K9 synthesis was maintained long-term. The results demonstrate that site-specific differentiation is an intrinsic property of postnatal human keratinocytes and can be expressed and maintained in a permissive environment in the absence of dermal tissue.