Effect of retinal and/or extra-retinal information on age in memory-guided saccades.

The aim of the study was to determine whether the addition of retinal or extra-retinal information could be used to improve memory-guided saccadic performance in healthy participants. Furthermore, we included two age groups in our study; healthy young adult subjects (mean age 20 years) and healthy middle-aged adult subjects (mean age 52 years). All subjects performed a novel task that incorporated a Go/NoGo task with a memory-guided saccade paradigm to investigate whether extra-retinal information (making a saccade towards the visible target) or retinal (a visible frame-of-reference) has any affect on the accuracy or variability of the response. We found all subjects made slight hypometric responses to the memory-guided targets. Both younger and middle-aged subjects revealed an increase in accuracy in the Go task compared with the NoGo task and the framed condition compared to the frameless condition, respectively. The frame also revealed a significant decrease in variability in the memory-guided saccades. A positive correlation in errors between the 1st and 2nd saccade in the Go task was revealed for all subjects; however, the older subjects revealed a greater correlation than younger subjects. The results presented indicate that younger and middle-aged perform highly similar patterns of errors during eye movements to remembered locations. However, middle-aged subjects show a greater tendency to use extra-retinal and retinal feedback to guide the response.

Identification of molecular markers linked to the mildew resistance gene Pl-d in apple.

Powdery mildew poses a serious problem for apple growers, and resistance to the disease is a major objective in breeding programmes for cultivar improvement. As selective pressure allows pathogens to overcome previously reliable resistances, there is a need for the introduction of novel resistance genes into new breeding lines. This investigation is concerned with the identification of the first set of molecular markers linked to the gene for mildew resistance, Pl-d, from the accession 'D12'. As no prior information on the map position or markers for Pl-d were available, a bulked-segregant approach was used to test 49 microsatellite primers, 176 amplified fragment length polymorphism (AFLP) primers and 80 random amplified polymorphic DNA (RAPD) primers in a progeny segregating for Pl-d resistance, 'Fiesta' (susceptible) x A871-14 ('Worcester Pearmain' x 'D12'). The segregations of the markers identified in the resistant and susceptible bulks were scored in the progeny, then the recombination fractions between Pl-d and the most tightly linked markers were calculated and a map prepared. Three AFLP, one RAPD and two microsatellite markers were identified. One AFLP was developed into a sequence-characterised amplified region marker, while the microsatellites CH03C02 and CH01D03 were flanking markers, 7 and 11 recombination units, respectively, from Pl-d. Two more distant microsatellites on the same linkage group, CH01D09 and CH01G12, confirmed the orientation of the markers on the linkage group. These microsatellites place Pl-d on the bottom of linkage group 12 in published apple maps, a region where a number of other disease resistance genes have been identified.

A non-IGF binding mutant of IGFBP-3 modulates cell function in breast epithelial cells.

We demonstrated previously that IGFBP-3 alone had no effect on cell death, but dramatically modulated apoptosis in Hs578T IGF non-responsive cells. We investigated whether a non-IGF binding mutant of IGFBP-3 retained its intrinsic actions in this cell line, prior to investigating its actions in IGF-responsive cells (MCF-7 and MCF-10A). In the Hs578T cells, the ceramide analogue, C2-induced apoptosis, non-glycosylated, glycosylated or mutant IGFBP-3 alone had no effect but on co-incubation with C2, all forms of IGFBP-3 markedly accentuated triggered apoptosis. In MCF-7 cells, IGFBP-3 was unable to modulate C2-induced death. In the MCF-10A cells, IGFBP-3 acted as a potent survival factor. IGFBP-3 also affected cell growth in the MCF-10A cells (inhibiting at low doses but increasing growth at higher concentrations). These actions of IGFBP-3 in the MCF-10A cells were independent of IGF-1. IGFBP-3 has differential IGF-independent effects on cell death and growth in normal breast and breast cancer cells.

Effects of a non-IGF binding mutant of IGFBP-5 on cell death in human breast cancer cells.

We have demonstrated previously that IGFBP-5 alone had no effect on cell death but modulated ceramide-induced apoptosis in Hs578T IGF non-responsive cells. To investigate if IGFBP-5 maintains its intrinsic ability to modulate apoptosis in IGF-responsive cells, we used a non-IGF binding mutant of IGFBP-5. In Hs578T cells, non-glycosylated, glycosylated or mutant IGFBP-5 alone each had no effect on cell death, whereas all forms inhibited ceramide-induced apoptosis. In IGF-responsive MCF-7 cells, each wild type form reduced ceramide-induced cell death but mutant IGFBP-5 was without effect. In the presence of mutant IGFBP-5, however, IGF-I no longer conferred survival and in the presence of wild type IGFBP-5, long R3 IGF-I was also unable to confer survival. In summary, all forms of IGFBP-5 modulated ceramide-induced apoptosis in Hs578T cells. In MCF-7 cells, IGF-I-induced survival could be facilitated by IGFBP-5, but also blocked by IGFBP-5 if association with IGFBP-5 was prevented.

Tissue transglutaminase facilitates the polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) and leads to loss of IGFBP-1's ability to inhibit insulin-like growth factor-I-stimulated protein synthesis.

Insulin-like growth factor-binding protein-1 (IGFBP-1) binds to insulin-like growth factors (IGFs) and has been shown to inhibit or stimulate cellular responses to IGF-I in vitro. This capacity of IGFBP-1 to inhibit or stimulate IGF-I actions correlates with its ability to form stable high molecular weight multimers. Since the ability of some proteins to polymerize is dependent upon transglutamination, we determined if tissue transglutaminase could catalyze this reaction and the effect of polymerization of IGFBP-1 upon IGF-I action. Following incubation with pure tissue transglutaminase (Tg), IGFBP-1 formed covalently linked multimers that were stable during SDS-polyacrylamide gel electrophoresis using reducing conditions. Dephosphorylated IGFBP-1 polymerized more rapidly and to a greater extent compared with native (phosphorylated) IGFBP-1. Exposure to IGF-I stimulated transglutamination of IGFBP-1 in vitro. An IGFBP-1 mutant in which Gln(66)-Gln(67) had been altered to Ala(66)-Ala(67) (Q66A/Q67A) was relatively resistant to polymerization by Tg compared with native IGFBP-1. Tg localized in fibroblast membranes was also shown to catalyze the formation of native IGFBP-1 multimers, however, Q66A/Q67A IGFBP-1 failed to polymerize. Although the mutant IGFBP-1 potently inhibited IGF-I stimulated protein synthesis in pSMC cultures, the same concentration of native IGFBP-1 had no inhibitory effect. The addition of higher concentrations of native IGFBP-1 did inhibit the protein synthesis response, and this degree of inhibition correlated with the amount of monomeric IGFBP-1 that was present. In conclusion, IGFBP-1 is a substrate for tissue transglutaminase and Tg leads to the formation of high molecular weight covalently linked multimers. Polymerization is an important post-translational modification of IGFBP-1 that regulates cellular responses to IGF-I.

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions.

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.

Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2.

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.

Hermeneutic analysis: a qualitative decision trail.

Qualitative analysis is problematic from two perspectives, which exist in the science and art debate [Tesch, R., 1990. Qualitative Research. Analysis types and software tools. The Falmer Press, London; Robson, C., 1993. Real World Research. Blackwell, Oxford]. The science view claims that the absence of clear and agreed analysis processes, which can be found in the quantitative domain, attracts labels to qualitative analysis of intuitive artistry and personal journeys which are considered 'unscientific'. This thinking remains dominant despite the growth of systematic qualitative analysis supported through computer analysis systems [Tesch, 1990]. However, in the art domain there is real resistance to the development of set methods of analysis which does view qualitative analysis, "as more of an art than a science" [Robson, 1993, p. 370]. This paper offers a contribution to the qualitative analysis tension through the promotion and illustration of a decision making trail. This option supports the principles of academic rigour in qualitative research [Guba, E.G., Lincoln, Y.S., 1981. Effective Evaluation. Jossey Bass, San Francisco] as a decision trail permits the research community to make their own judgements concerning the process of analysis, the overall trustworthiness of the research and therefore its presented interpretations.

Evidence-based practice: a retrograde step? The importance of pluralism in evidence generation for the practice of health care.

The origin of evidence-based medicine is explored and its connection to evidence-based practice examined. Widespread acceptance of the dominant scientific paradigm supporting evidence-based practice is challenged. Knowledge classifications and their relevance for nursing practice are considered, with the need for a critical balance to be pursued. Recommendations are made, regarding the future of evidence for practice, and a brief example of emerging evidence concerning values of professional caring is offered in redressing the balance.

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions.

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.

Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.

Protease-resistant form of insulin-like growth factor-binding protein 5 is an inhibitor of insulin-like growth factor-I actions on porcine smooth muscle cells in culture.

IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture. The effects of IGFs on cells are modulated by various insulin-like growth factor-binding proteins (IGFBP). IGFBP-5 is synthesized by pSMC and binds to the extracellular matrix. However, IGFBP-5 is also secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact IGFBP-5. To study the consequence of accumulation of intact IGFBP-5 in medium, we determined the cleavage site in IGFBP-5 and prepared a protease resistant mutant. Amino acid sequencing of purified IGFBP-5 fragments suggested Arg138-Arg139 as the primary cleavage site. Arg138-Arg139-->Asn138-Asn139 mutations were introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein. This mutant IGFBP-5 remained intact even after 24 h of incubation and it inhibited several IGF-I actions when added to pSMC culture medium. The mutant IGFBP-5 (500 ng/ml) decreased IGF-I stimulated cellular DNA synthesis by 84%, protein synthesis by 77%, and it inhibited IGF-I stimulated migration of pSMC by 77%. It also inhibited IGF-I stimulation of IRS-1 phosphorylation. In contrast, the same amount of native IGFBP-5 did not inhibit IGF-I actions. The significance of inhibitory effects of the protease resistant IGFBP-5 was further demonstrated in pSMC transfected with mutant or native IGFBP-5 cDNAs. The mutant IGFBP-5 accumulated in culture medium of transfected cells, while native IGFBP-5 was degraded into fragments, PSMC overexpressing the mutant IGFBP-5 also responded poorly to IGF-I compared with mock transfected cells. IGF-I (5 ng/ml) increased [35S]methionine incorporation into control cells by 36% above the basal level, but it did not significantly change (4%) in pSMC cultures that were producing the mutant IGFBP-5. In conclusion, the accumulation of protease-resistant IGFBP-5 in the medium was inhibitory to IGF-I actions on pSMC. This suggests that proteolysis can prevent IGFBP-5 from acting as an inhibitor of IGF-I-stimulated effects and that it serves as an important mechanism for regulating cellular responsiveness to IGF-I.

Academic validation of prior and experiential learning: evaluation of the process.

This report is a summary of an AP(E)L evaluative research project conducted at the University of Southampton School of Nursing and Midwifery. The project aimed to shed light on the perceptions of candidates and teachers in conjunction with the effectiveness of the accreditation process. Through initial semi-structured interviews with teachers (n = 6) and candidates (n = 6) offering their perceptions and experiences, an emergent questionnaire was sent to the remaining active accreditation candidates (n = 60, response n = 39) and advisers/assessors (n = 36), response n = 32). The effectiveness of candidate preparation for beginning an accreditation claim, the comprehensiveness of the types of credit available to capture learning through experience, the styles of advising and feedback from assessment are all reported on. Interestingly, the data highlight how preparing an accreditation claim can affect both adviser and candidate, in terms of being more self-critical and reflective in their areas of practice.

Detection of human viruses using primary cells immortalised by oncogene transfection, in comparison with primary cells and established cell lines.

No single established cell line was found capable of substituting for primary baboon kidney (PBK) or primary rhesus macacque kidney (PRK) cells for detection of human viruses. Although a panel of cell lines could detect influenza, parainfluenza, and enteroviruses, which are among the most important viruses encountered in routine diagnostic laboratories, the sensitivity of this panel was not as high as that of PBK or PRK cells. However, in a promising complementary approach, PBK and PRK cells have been immortalised successfully by oncogene transfection, and some of the resulting cell lines have retained susceptibility to human viruses, and may be suitable for routine diagnostic use.

Identification of the extracellular matrix binding sites for insulin-like growth factor-binding protein 5.

Fibroblast extracellular matrix (ECM) contains two forms of insulin-like growth factor-binding proteins (IGFBPs), IGFBP-3 and IGFBP-5. These studies were undertaken to identify the regions within IGFBP-5 that mediate its binding to fibroblast ECM. Synthetic peptides were prepared that were homologous with two regions of basic amino acids within IGFBP-5 (Arg201-Arg218 and Ala131-Thr141). Increasing concentrations of both peptides competed with IGFBP-5 for binding to ECM but the Arg201-Arg218 peptide was more potent. Mutagenesis was used to define the effect of substituting for these basic residues on ECM binding. Substitution for two peptide B residues K134A and R136A reduced binding by 40%. Substitution of a single basic residue within the peptide A region (K211N) reduced binding to ECM by 49%. Substitution for K211N, K134A, and R136A reduced binding by 52%. More extensive substitutions in the peptide A region, e.g. K211N,R214A,K217A,R218N, resulted in a greater (e.g. 88%) decrease. The positional location of basic residues appeared to be more important than the total number of substitutions since the mutant K202N,K206A,R207A had a 79% reduction in ECM binding. Two basic regions of IGFBP-5 contribute to its binding to ECM, but the region containing amino acids 201-218 has a greater contribution. ECM binding is mediated by charged residues and acts to stabilize IGFBP-5 by protecting it from proteolysis.

Halogenated anesthetics form liver adducts and antigens that cross-react with halothane-induced antibodies.

Two halogenated anesthetics, enflurane and isoflurane, have been associated with an allergic-type hepatic injury both alone and following previous exposure to halothane. Halothane hepatitis appears to involve an aberrant immune response. An antibody response to a protein-bound biotransformation product (trifluoroacetyl adduct) has been detected on halothane hepatitis patients. This study was performed to determine cross-reactivity between enflurane and isoflurane with the hypersensitivity induced by halothane. The subcellular and lobular production of hepatic neoantigens recognized by halothane-induced antibodies following enflurane and isoflurane, and the biochemical nature of these neoantigens was investigated in two animal models. Enflurane administration resulted in neoantigens detected in both the microsomal and cytosolic fraction of liver homogenates and in the centrilobular region of the liver. In the same liver, biochemical analysis detected fluorinated liver adducts that were up to 20-fold greater in guinea pigs than in rats. This supports and extends previous evidence for a mechanism by which enflurane and/or isoflurane could produce a hypersensitivity condition similar to that of halothane hepatitis either alone or subsequent to halothane administration. The guinea pig would appear to be a useful model for further investigations of the immunological response to these antigens.

Turning experience into academic learning. The APL and APEL credit systems.

1. Learning from experience can be turned into academic credit through APL and APEL. 2. The APL/APEL process is about valuing and empowering practitioners. 3. Reflection is central to developing an APEL claim. 4. An accreditation model offers a five-stage process leading to the award of credit.

Role of insulin-like growth factor binding proteins in the control of IGF actions.

The insulin-like growth factor binding proteins have been shown to modify IGF actions. IGFBP-5 binds to extracellular matrix (ECM) and its ability to potentiate IGF activity is dependent upon the amount that is ECM associated. To determine the specific regions of IGFBP-5 that are required for ECM association, site directed mutagenesis has been used to prepare several forms of IGFBP-5. Mutants that have had the amino acids between positions 201 and 218 altered have been useful. Mutation of the lysine 211 resulted in no change in the affinity of IGFBP-5 for ECM or heparin Sepharose; however, it resulted in a major reduction in affinity for IGF-I following heparin binding. Other mutations which disrupted heparin binding also resulted in loss of this affinity shift. Most distruptive were mutations of amino acids 211, 214, 217 and 218 and 202, 206 and 207. Mutation of residues 201 plus 202 had some effect, but substitution for 207, 211, 217 and 218 had no effect. When binding to intact ECM was analyzed, similar results were obtained. This suggests that amino acids 202, 206 and 214 are definitely involved in heparin and ECM binding. When binding to proteoglycans such as tenascin and heparin sulfate proteoglycan was analyzed, similar results were obtained. IGFBP-5 also binds to other proteins in ECM, including type IV collagen and plasminogen activator inhibitor-I. Specific antisera for plasminogen activator inhibitor-1 can coprecipitate IGFBP-5. IGFBPs are degraded by specific proteases. Three proteases that degrade IGFBP-2, -4 and -5 have been characterized. They are serine proteases that cleave these proteins at basic residues. Although several well characterized serine proteases cleave IGFBP-4 or -5, the proteases in cell conditioned media appear to be distinct.

CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.