Sterilization of a new medical device using broad-spectrum pulsed light.

In the development of implantable medical devices, effective sterilization is an essential design element. This article outlines how broad-spectrum pulsed light (BSPL) has been implemented to sterilize a novel, implantable medical device. Components of the device have properties incompatible with most sterilization techniques. The unique characteristics of the device and sterilization method are described. Results are presented that show BSPL can be an effective sterilization method that has the potential to meet validation requirements to allow parametric release of the treated product.

Pit-1 binding to specific DNA sites as a monomer or dimer determines gene-specific use of a tyrosine-dependent synergy domain.

Transcriptional activation of the prolactin and growth hormone genes, occurring in a cell-specific fashion, requires short-range synergistic interactions between the pituitary-specific POU domain factor Pit-1 and other transcription factors, particularly nuclear receptors. Unexpectedly, we find that these events involve the gene-specific use of alternative Pit-1 synergy domains. Synergistic activation of the prolactin gene by Pit-1 and the estrogen receptor requires a Pit-1 amino-terminal 25-amino-acid domain that is not required for analogous synergistic activation of the growth hormone promoter. The action of this Pit-1 synergy domain is dependent on the presence of two of three tyrosine residues spaced by 6 amino acids and can be replaced by a comparable tyrosine-dependent trans-activation domain of an unrelated transcription factor (hLEF). The gene-specific utilization of this tyrosine-dependent synergy domain is conferred by specific Pit-1 DNA-binding sites that determine whether Pit-1 binds as a monomer or a dimer. Thus, the critical DNA site in the prolactin enhancer, where this domain is required, binds Pit-1 as a monomer, whereas the Pit-1 sites in the growth hormone gene, which do not utilize this synergy domain, bind Pit-1 as a dimer. The finding that the sequence of specific DNA sites dictates alternative Pit-1 synergy domain utilization based on monomeric or dimeric binding suggests an additional regulatory strategy for differential target gene activation in distinct cell types.

Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site.

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors.

Characterization of several thyroid hormone (T3), retinoic acid, and estrogen response elements has led to the identification of conserved DNA half-sites (core binding motifs). We present evidence that differences in both the relative orientation and spacing of these motifs within hormone response elements determine the distinct transcriptional responses of three members of the nuclear receptor superfamily. When separated by 3 bp, direct repeat, palindromic, and inverted palindromic arrangements of these motifs impart selective transcriptional responses to retinoic acid, estrogen, and T3 receptors, respectively. Varying the spacing between core motifs alters the specificity. Without spacing, a direct repeat of the core motif paradoxically configures the T3 receptor to confer transactivation in the absence of T3 and repression in its presence. Such an element occurs naturally in the mouse beta-thyrotropin promoter, physiologically under negative regulation by T3. The orientation and spacing of core binding motifs may thus function in concert as a code that accounts for the selective patterns of transcriptional responses of hormonally regulated promoters.

The C'-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity.

To investigate mechanisms responsible for positive and negative transcriptional control, we have utilized two types of promoters that are differentially regulated by thyroid hormone (T3) receptors. Promoters containing the palindromic T3 response element TCAGGTCA TGACCTGA are positively regulated by the T3 receptor after the administration of T3, whereas otherwise identical promoters containing the estrogen response element TCAGGTCA CTG TGACCTGA can be regulated negatively; converse effects are observed with the estrogen receptor. We describe evidence that the transcriptional inhibitory effects of the T3 or estrogen receptors on the estrogen or T3 response elements, respectively, are imposed by amino acid sequences in the C'-terminal region that colocalize with dimerization and hormone-binding domains and that these sequences can transfer inhibitory functions to other classes of transcription factors. Removal of the C'-terminal dimerization and hormone-binding domains of either the alpha T3 or estrogen receptors permits each receptor to act constitutively to enhance transcription on both T3 and estrogen response elements. It is, therefore, suggested that protein-protein interactions between receptor C' termini limit the subset of DNA binding sites on which transcriptional activation occurs.

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-1 protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-1, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-1, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.

The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements.

We report that the thyroid hormone (T3) receptor binds to DNA recognition sequences that are variations of the palindromic motif 5'-TCAGGTCA.TGACCTGA-3', resulting in transcriptional activation in vivo. This sequence is identical to the vitellogenin A2 estrogen response element except for the absence of 3 bp at the center of dyad symmetry, which we have termed a "gap." The T3 receptor binds to estrogen response elements with high affinity in vitro, as well as to variations of the same sequence containing gaps of 1-6 bp. The T3 receptor not only fails to activate transcription from estrogen response elements in vivo, but inhibits estrogen-dependent transactivation. We propose that the T3 receptor binds to estrogen response elements in a transcriptionally inactive form and competes for estrogen receptor binding, resulting in a net decrease in gene expression. These data reveal that only a subset of T3 receptor DNA-binding elements function as T3 response elements.

Inhibition of human natural killer cell activity by platelet-derived growth factor.

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.