Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to ≈9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein (≈68 kDa). tRA stimulated NIS gene transcription ≈4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t1/2 = 24 min), compared with that in FRTL-5 thyroid cells (t1/2 = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with 131I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness

The human β2-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5′ upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common β2-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to β agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. β2-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were ≈50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI–RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

A method for directed evolution and functional cloning of enzymes

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold, P < 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1+lin− adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- ± 2-fold, myeloid colony-forming cells increased 140- ± 36-fold, and total nucleated cells increased 230- ± 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1+lin− marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119− CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.

Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells

Previous studies have demonstrated hematopoietic stem cell amplification in vitro after the activation of three cell-surface receptors: flt3/flk2, c-kit, and gp130. We now show flt3-ligand and Steel factor alone will stimulate >85% of c-kit+Sca-1+lin− adult mouse bone marrow cells to proliferate in single-cell serum-free cultures, but concomitant retention of their stem cell activity requires additional exposure to a ligand that will activate gp130. Moreover, this response is restricted to a narrow range of gp130-activating ligand concentrations, above and below which hematopoietic stem cell activity is lost. These findings indicate a unique contribution of gp130 signaling to the maintenance of hematopoietic stem cell function when these cells are stimulated to divide with additional differential effects dictated by the intensity of gp130 activation.

The effects of aging on gene expression in the hypothalamus and cortex of mice

A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C β, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ were significantly enhanced. Hyperphosphorylated τ is the major constituent of the neurofibrillary tangles in the brains of Alzheimer's disease patients. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-β sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.

SMART, a simple modular architecture research tool: Identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387–392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Web-based distance continuing education: a new way of thinking for students and instructors

As people have more difficulty taking time away from work to attend conferences and workshops, the idea of offering courses via the Web has become more desirable. Addressing a need voiced by Medical Library Association membership, the authors developed a Web-based continuing-education course on the subject of the librarian's role in evidence-based medicine. The aim of the course was to provide medical librarians with a well-constructed, content-rich learning experience available to them at their convenience via the Web. This paper includes a discussion of the considerations that need to be taken into account when developing Web-based courses, the issues that arise when the information delivery changes from face-to-face to online, the changing role of the instructor, and the pros and cons of offering Web-based versus traditional courses. The results of the beta test and future plans for the course are also discussed.