Parental Expressed Concerns about Silver Diamine Fluoride (SDF) Treatment.

Background: The staining associated with its caries arrest may be a deterrent for the use of Silver Diamine Fluoride (SDF). This study aims to elucidate the concerns that inform parents' perceptions and acceptance of SDF as a treatment option for their child. Study Design: We analyzed qualitative data obtained through an investigation in which parents attending a pediatric dental appointment participated in a survey, which included an open-ended question to evaluate their opinions about SDF staining. Thematic analysis of the comments, offered by the subsample of participants who replied to this question (n=43), yielded insights about perception of SDF therapy. Results: Most parents who provided comments were mothers (83.7%), college graduates (72.1%), primarily white (48.8%) or Hispanic (27.9%). Six themes emerged from the thematic analysis of the parents' responses: Esthetic Concerns, Psychosocial Concerns, SDF Treatment Process, Risks and Side Effects, Situational Benefits, and Dental Treatment Process. While many of the parents' comments are related to appearance, other topics that merit consideration when discussing SDF treatment were mentioned. Conclusions: Although parents are concerned about the esthetic impact of SDF, they understand the risks of alternative treatments and welcome information that will allow them to make an informed decision. Location of the cavities and visibility of the staining appear to heavily influence the decision to accept or reject this therapy.

In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries.

The covalent coupling of an mRNA to the protein that it encodes (mRNA display) provides a powerful tool for analysis of protein function in the post-genomic era. This coupling allows the selective enrichment of individual members from libraries of displayed proteins and the subsequent regeneration of an enriched library using the RNA moiety. Tissue-specific libraries from poly(A)(+) mRNA were prepared by priming first and second strand cDNA synthesis with oligonucleotides containing nine random 3' nucleotides, the fixed regions of which encoded the requisite sequences for formation of mRNA display constructs and a library-specific sequence tag. Starting with a pool of uniquely tagged libraries from different tissues, an iterative selection was performed for binding partners of the anti-apoptotic protein Bcl-X(L). After four rounds of selection, the pool was deconvoluted by polymerase chain reaction amplification with library-specific primers. Subsequent clonal sequence analysis revealed the selection of three members of the Bcl-2 family known to bind to Bcl-X(L). In addition, several proteins not previously demonstrated to interact with Bcl-X(L) were identified. The relative binding affinities of individual selected peptides were determined, as was their susceptibility to competition with a BH3 domain peptide. Based on these data, a putative BH3 domain was identified in most peptides.

Analysis of eosinophils and myeloid progenitor responses to modified forms of MPIF-2.

Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.

PROfusion: genetically tagged proteins for functional proteomics and beyond.

As a new enabling technology in functional genomics, Phylos, Inc. has pioneered PROfusiontrade mark technology, whereby proteins are covalently tagged with their own genetic information (mRNA). Using this technology, both synthetic and natural libraries (representing the repertoire of proteins naturally expressed in a given cell type or tissue source) have been constructed. Due to the in vitro nature of library construction, Phylos libraries are the largest described to date, up to 10(14) in size. From a library of such molecules, one can select for a protein function of choice with the benefit that the genetic material is linked to the protein for subsequent PCR amplification, enrichment, and, ultimately, identification.

Identification and characterization of a new human ETS-family transcription factor, TEL2, that is expressed in hematopoietic tissues and can associate with TEL1/ETV6.

The ETS family of proteins is a large group of transcription factors implicated in many aspects of normal hematopoietic development, as well as oncogenesis. For example, the TEL1/ETV6 (TEL1) gene is required for normal yolk sac angiogenesis, adult bone marrow hematopoiesis, and is rearranged or deleted in numerous leukemias. This report describes the cloning and characterization of a novel ETS gene that is highly related to TEL1 and is therefore called TEL2. The TEL2 gene consists of 8 exons spanning approximately 21 kilobases (kb) in human chromosome 6p21. Unlike the ubiquitously expressed TEL1 gene, however, TEL2 appears to be expressed predominantly in hematopoietic tissues. Antibodies raised against the C-terminus of the TEL2 protein were used to show that TEL2 localizes to the nucleus. All ETS proteins can bind DNA via the highly conserved ETS domain, which recognizes a purine-rich DNA sequence with a GGAA core motif. DNA binding assays show that TEL2 can bind the same consensus DNA binding sequence recognized by TEL1/ETV6. Additionally, the TEL2 protein is capable of associating with itself and with TEL1 in doubly transfected Hela cells, and this interaction is mediated through the pointed (PNT) domain of TEL1. The striking similarities of TEL2 to the oncogenic TEL1, its expression in hematopoietic tissues, and its ability to associate with TEL1 suggest that TEL2 may be an important hematopoietic regulatory protein.

Dendritic cells and MPIF-1: chemotactic activity and inhibition of endogenous chemokine production by IFN-gamma and CD40 ligation.

We have examined the biological activity of the CC chemokine myeloid progenitor inhibitory factor 1 (MPIF-1) on human dendritic cells. MPIF-1 has chemotactic activity on dendritic cells derived from either peripheral blood monocytes or cord blood CD34+ progenitors. However, chemokine treatment did not induce further cell activation or maturation. In addition, MPIF-1 is constitutively released by monocyte-derived dendritic cells but not macrophages or monocytes (resting or stimulated). The proinflammatory stimuli lipopolysaccharide and tumor necrosis factor alpha, which induced the release of monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and interleukin-8, did not affect MPIF-1 release. In contrast, CD40 ligation and interferon-gamma treatment, while stimulating the production of the other chemokines, caused a pronounced reduction of MPIF-1 transcript and protein release. Thus, in dendritic cells the regulation of the production and release of MPIF-1 is distinct in comparison to other CC and CXC chemokines.

STRL22 is a receptor for the CC chemokine MIP-3alpha.

STRL22 is a human seven transmembrane domain orphan receptor related to known chemokine receptors and expressed in peripheral blood lymphocytes, tumor infiltrating lymphocytes and lymphoid tissues. MIP-3alpha/LARC/Exodus is a CC chemokine that is chemotactic for lymphocytes and that is expressed in activated cells, including monocytes, T cells, endothelial cells, and fibroblasts, and in liver, lung, and some lymphoid tissues. We report here that STRL22-transfected human embryonic kidney 293 cells demonstrated specific binding for MIP-3alpha and that MIP-3alpha, but no other chemokines, produced a calcium flux in the STRL22-transfected cells. We show that MIP-3alpha, unlike other chemokines, produced a calcium flux in freshly-isolated peripheral blood lymphocytes and we show that MIP-3alpha also produced a signal in tumor infiltrating lymphocytes that express STRL22. Since STRL22 is the sixth functional CC chemokine receptor identified, it should be re-named CCR6.

Quantification of the effects of astrocytes on oligodendroglial morphology.

Oligodendrocytes in neonatal rat forebrain cultures grow either in isolation of other cells or upon astrocytes. Populations of both types of oligodendrocytes were used to quantify the effects of astrocytes on oligodendroglial morphology. Changes in oligodendroglial size and shape were determined by measurement of total process length, cell area, growth area, and fractal dimension. The directionality of process growth, quantified by measurement of the axes of growth, was also compared. Isolated oligodendrocytes exhibited greater total process length, greater cellular area, larger growth area, and a more complex boundary than oligodendrocytes growing upon astrocytes. Analysis of the axes of cellular growth revealed that the processes of isolated oligodendrocytes exhibited radial symmetry, whereas the processes of oligodendrocytes growing upon astrocytes were limited to an area demarcated by the astrocytic processes. These data suggest that, in neuron-free culture, the growth of oligodendroglial processes is modified by underlying astrocytic processes.

Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

Basic fibroblast growth factor prevents cAMP-induced apoptosis in cultured Schwann cells.

Peripheral nerve axotomy induces apoptosis in Schwann cell precursors; basic fibroblast growth factor (bFGF) protects these cells from axotomy-induced apoptosis (Jessen et al.: Neuron 12:509-527, 1994; Gavrilovic et al.: Eur J Neurosci 7:7-85, 1995). In this study, we investigate the effects of bFGF on apoptosis in neuron-free cultures of neonatal rat Schwann cells. Apoptotic cell death was induced in primary and secondary expanded Schwann cells by treatment with 1 mM concentrations of 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP), a membrane-permeable analogue of cAMP which induces expression of galactocerebroside in the plasma membranes of Schwann cells. Treatment with bFGF reduced the percentage of galactocerebroside-bearing Schwann cells undergoing cAMP-induced DNA fragmentation. These findings suggest that bFGF can enhance the survival of terminally differentiated Schwann cells by preventing apoptosis.

Health insurance and the homeless.

There is very little known about health care utilization among the homeless or about the role of health insurance on utilization patterns. Many health care reform proposals advocate expanding health insurance coverage for various segments of society, including the homeless. Although homeless people who lack health insurance face strong financial barriers to health services, providing them with health insurance may not appreciably increase their demand for health care if they also face important non-financial barriers. We investigate the relationship between insurance and utilization for this group based on estimates from an empirical model of medical care use and insurance coverage. Using our estimates, we simulate potential effects of policy changes on various types of utilization, including use of mental health services and treatment for alcohol or other drug abuse.

Complexity analysis of oligodendroglial processes expressing myelin-associated glycoprotein.

Oligodendroglia synthesize myelin in the mammalian central nervous system. Mature oligodendroglia have been identified in culture by two criteria; the expression of molecules characteristic of myelin, such as galactocerebroside (galC) and myelin-associated glycoprotein (MAG), and the elaboration of complex processes. Myelin gene expression can be documented by the binding of specific antibodies and antisera to the myelin-specific molecules; process complexity can be described by the fractal dimension, D. In this study, anti-MAG antisera was used to document MAG expression in the processes of oligodendroglia. Eighty percent of the galC+ oligodendroglia bound anti-MAG antiserum. With time in culture, MAG immunoreactivity seemed to extend from the cell soma into the oligodendroglial processes. To quantify this observation, fractal dimensions were calculated using either galC or MAG immunoreactivity to visualize oligodendroglial processes. A fractal dimension of 1.5 was calculated for O1+ processes by day 4 of culture; this value for D remained constant over the course of 1 month in culture. The fractal dimension calculated for MAG+ processes increased from 1.2 to 1.5 over the course of 28 days in culture. This change in fractal dimension confirms our visual impression that galC-containing processes acquire MAG slowly over the course of several weeks in culture.

Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

Myelin gene expression in glia treated with oligodendroglial trophic factor.

Oligodendroglia synthesize myelin in the CNS. In vitro, oligodendroglia may be identified by the binding of monoclonal antibodies against galactocerebroside, a myelin-specific galactolipid. Oligodendroglial trophic factor is a protein mitogen for cells of the oligodendroglial lineage. When oligodendroglia in cerebral white matter cultures are treated with oligodendroglial trophic factor, galactocerebroside-positive cells undergo mitosis but fail to express the myelin structural proteins, myelin basic protein and proteolipid protein. Oligodendroglia treated with oligodendroglial trophic factor, however, do express 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein in a manner similar to oligodendroglia treated with platelet-derived growth factor. Oligodendroglial trophic factor, therefore, generates a population of somewhat 'immature' oligodendroglia, which are galactocerebroside, myelin-associated glycoprotein and 2', 3'-cyclic nucleotide 3' phosphodiesterase positive but myelin basic protein and proteolipid protein negative.

The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences.

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.

Interleukin-3 signals through multiple isoforms of Stat5.

The interleukin (IL)-3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL-3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL-3 activates STF-IL-3a, which comprises two tyrosine-phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL-3 and granulocyte-macrophage colony-stimulating factor activate STF-IL-3b, which consists of a 94 and 96 kDa tyrosine-phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF-IL-3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL-3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.

Partial purification of a novel mitogen for oligodendroglia.

A protein with a MWapp of 50-70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS-polyacrylamide gel, this protein ("oligodendroglial trophic factor," OTF) elicited half-maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet-derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage.

Cytokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors.

The ability of cytokines to activate distinct but overlapping sets of genes defines their characteristic biological response. We now show that IFN gamma, IL-3, IL-4, IL-6, erythropoietin, EGF, and CSF-1 activate differing members of a family of latent cytoplasmic transcription factors. Although these factors have distinct physical and functional properties and exhibit different patterns of expression, they share many important features, including recognition of a related set of enhancer elements, rapid activation, tyrosine phosphorylation, and cross-reactivity to antibodies against p91, a cytoplasmic signaling protein activated by IFN alpha, IFN gamma, and IL-6. These shared features point to either parallel or common patterns of signal transduction. A general model of cytokine signal transduction is presented, in which receptor-associated tyrosine kinases activate ligand-specific members of a family of signal-transducing factors. Once activated, these factors carry their signals to the nucleus, where they bind a family of related enhancer elements.

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

The carboxyl domain of zinc fingers of the Evi-1 myeloid transforming gene binds a consensus sequence of GAAGATGAG.

Inappropriate expression of the Evi-1 zinc finger gene is associated with myeloid leukemia and myelodysplastic syndromes in mice and humans and has been hypothesized to contribute to pathology by blocking myeloid differentiation. Evi-1 contains two domains of zinc fingers, an amino-terminal domain of seven fingers and a carboxyl domain of three fingers. The first domain binds a consensus sequence of GA(C/T)AAGATAAGATAA in binding and amplication reactions or GATA repeat containing regions of genomic DNA. The experiments described here, establish a consensus sequence for the carboxyl domain of zinc fingers consisting of GAAGATGAG. Unlike the first domain, the consensus sequence established for the carboxyl domain is identical to that which would be predicted by the current rules relating to C2H2 zinc fingers and DNA recognition. Substitution of sequences in finger 8 with those in finger 9, demonstrate that the individual fingers bind the predicted region of the consensus sequence. In an attempt to engineer binding of constructs containing the carboxyl domain, a variety of mutations were made in the middle finger that would be predicted to change the consensus sequence in specific ways. Remarkably, most of the mutations were deleterious and destroyed specific DNA binding. Although Evi-1 contains potential transcriptional activation domains, it was not able to activate gene transcription from CAT constructs containing the consensus sequence.