The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V(max) and K(m), as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P(dif)). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V(max) and K(m) were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5-6-fold higher permeability at 37 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.

We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer.

Vascular endothelial growth factor (VEGF) is a cytokine with main angiogenetic functions in embryonic development and tumor-formation. In the adult lung, reports of the localization of VEGF were controversial. A precise cell typing of VEGF-positive pulmonary cells is still lacking. Nothing is known about a potential role in pulmonary fibrosis. Immunohistochemistry (IH), double immunofluorescence microscopy (DIF), and immunoelectron microscopy (IEM) were used to study the differential distribution of VEGF in paraffin-embedded (IH, DIF) and in cryo-substituted, Lowicryl-embedded (IEM) specimens of normal rat and human lungs and fibrotic rat lungs. Fibrosis was induced by intratracheal bleomycin treatment. IH and DIF showed that VEGF was present in surfactant protein (SP) D-positive alveolar type II pneumocytes, bronchiolar Clara cells, smooth muscle (SM) cells, and alpha-SM actin-positive myofibroblasts of normal rat and human lungs. Fibrotic lesions in bleomycin-treated rat lungs were rich in VEGF-positive cells presenting with a heterogeneous phenotype (mainly SP-D-positive type II pneumocytes, alpha-SM actin-positive myofibroblasts). There were no signs of angiogenesis. Post-embedding immunogold labeling using protein A-gold and IgG-gold technique revealed a specific localization of VEGF to mitochondria, Clara cell secretory granules, and capillary interendothelial cell junctions. The predominant localization of VEGF to bronchiolar and alveolar epithelial and alpha-SM actin-positive cells, and the marked increase of VEGF-positive type II pneumocytes and myofibroblasts in fibrotic lung lesions, indicate that in adult lungs VEGF is involved in processes other than angiogenesis.

To investigate how cell type proportions are regulated during Dictyostelium development, we have attempted to find out which cell type produces DIF-1, a diffusible signal molecule inducing the differentiation of prestalk-O cells. DIF-1 is a chlorinated alkyl phenone that is synthesized from a C12 polyketide precursor by chlorination and methylation, with the final step catalysed by the dmtA methyltransferase. All our evidence points to the prespore cells as the major source of DIF-1. (1) dmtA mRNA and enzyme activity are greatly enriched in prespore compared with prestalk cells. The chlorinating activity is also somewhat prespore-enriched. (2) Expression of dmtA is induced by cyclic-AMP and this induction is inhibited by DIF-1. This regulatory behaviour is characteristic of prespore products. (3) Short-term labelling experiments, using the polyketide precursor, show that purified prespore cells produce DIF-1 at more than 20 times the rate of prestalk cells. (4) Although DIF-1 has little effect on its own synthesis in short-term labelling experiments, in long-term experiments, using 36Cl(-) as label, it is strongly inhibitory (IC(50) about 5 nM), presumably because it represses expression of dmtA; this is again consistent with DIF-1 production by prespore cells. Inhibition takes about 1 hour to become effective. We propose that prespore cells cross-induce the differentiation of prestalk-O cells by making DIF-1, and that this is one of the regulatory loops that sets the proportion of prespore-to-prestalk cells in the aggregate.

OBJECTIVE

To examine the measurement equivalence of items on disability across three international surveys of aging.

METHODS

Data for persons aged 65 and older were drawn from the Health and Retirement Survey (HRS, n = 10,905), English Longitudinal Study of Aging (ELSA, n = 5,437), and Survey of Health, Ageing and Retirement in Europe (SHARE, n = 13,408). Differential item functioning (DIF) was assessed using item response theory (IRT) methods for activities of daily living (ADL) and instrumental activities of daily living (IADL) items.

RESULTS

HRS and SHARE exhibited measurement equivalence, but 6 of 11 items in ELSA demonstrated meaningful DIF. At the scale level, this item-level DIF affected scores reflecting greater disability. IRT methods also spread out score distributions and shifted scores higher (toward greater disability). Results for mean disability differences by demographic characteristics, using original and DIF-adjusted scores, were the same overall but differed for some subgroup comparisons involving ELSA.

CONCLUSIONS

Testing and adjusting for DIF is one means of minimizing measurement error in cross-national survey comparisons. IRT methods were used to evaluate potential measurement bias in disability comparisons across three international surveys of aging. The analysis also suggested DIF was mitigated for scales including both ADL and IADL and that summary indexes (counts of limitations) likely underestimate mean disability in these international populations.

Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.

OBJECTIVE

Differential item functioning (DIF) analyses are increasingly used to evaluate health-related quality of life (HRQoL) instruments, which often include relatively short subscales. Computer simulations were used to explore how various factors including scale length affect analysis of DIF by ordinal logistic regression.

METHODS

Simulated data, representative of HRQoL scales with four-category items, were generated. The power and type I error rates of the DIF method were then investigated when, respectively, DIF was deliberately introduced and when no DIF was added. The sample size, scale length, floor effects (FEs) and significance level were varied.

RESULTS

When there was no DIF, type I error rates were close to 5%. Detecting moderate uniform DIF in a two-item scale required a sample size of 300 per group for adequate (>80%) power. For longer scales, a sample size of 200 was adequate. Considerably larger sample sizes were required to detect nonuniform DIF, when there were extreme FEs or when a reduced type I error rate was required.

CONCLUSIONS

The impact of the number of items in the scale was relatively small. Ordinal logistic regression successfully detects DIF for HRQoL instruments with short scales. Sample size guidelines are provided.

Little is known about the psychometric properties of depression instruments among persons infected with HIV. We analyzed data from a large sample of patients in usual care in two US cities (n=1467) using the nine-item Patient Health Questionnaire (PHQ-9) from the PRIME-MD. The PHQ-9 had curvilinear scaling properties and varying levels of measurement precision along the continuum of depression measured by the instrument. In our cohort, the scale showed a prominent floor effect and a distribution of scores across depression severity levels. Three items had differential item functioning (DIF) with respect to race (African-American vs. white); two had DIF with respect to sex; and one had DIF with respect to age. There was minimal individual-level DIF impact. Twenty percent of the difference in mean depression levels between African-Americans and whites was due to DIF. While standard scores for the PHQ-9 may be appropriate for use with individual HIV-infected patients in cross-sectional settings, these results suggest that investigations of depression across groups and within patients across time may require a more sophisticated analytic framework.

OBJECTIVE

Accurate assessment of racial disparities in attention-deficit/hyperactivity disorder (ADHD) depends on measurement that is equally valid for all groups. This study examines differences among African American and white children in ADHD measurement with a widely used parental report instrument, the Diagnostic Interview Schedule for Children (DISC).

METHODS

Data come from 1070 children in the Fast Track Project, a longitudinal study of predominantly low-income children at risk of emotional and/or behavioral problems. Item Response Theory (IRT) methodology is used to determine whether ADHD screening items provide comparable information for African American and white children or whether differential item function (DIF) exists. IRT scores and race/ethnicity are entered in logistic regression models predicting use of ADHD medication.

RESULTS

Seven of 39 DISC items performed differently among African Americans and whites. In most cases, parents of white children were more likely to endorse these items than were parents of African American children at comparable underlying levels of children's hyperactivity. When items exhibiting differential functioning were deleted, race disparities predicting underlying need as indicated by ADHD medication use decreased and were no longer statistically significant.

CONCLUSIONS

Perceptions of ADHD-related symptoms among parents of African American children appear to differ in important ways from those of parents of white children, and screening instruments relying on parent report may yield different results for African American and white children with similar underlying treatment needs. Gathering information from additional sources including teachers and school counselors can provide a more complete picture of the behavioral functioning and therapeutic needs of children in all race/ethnic groups.

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

BACKGROUND

The aim of this study was to test the internal validity of the total Center for Epidemiologic Studies-Depression (CES-D) scale using Rasch analysis in a rheumatoid arthritis (RA) population.

METHODS

CES-D was administered to 157 patients with RA over three time points within a 12 month period. Rasch analysis was applied using RUMM2020 software to assess the overall fit of the model, the response scale used, individual item fit, differential item functioning (DIF) and person separation.

RESULTS

Pooled data across three time points was shown to fit the Rasch model with removal of seven items from the original 20-item CES-D scale. It was necessary to rescore the response format from four to three categories in order to improve the scale's fit. Two items demonstrated some DIF for age and gender but were retained within the 13-item CES-D scale. A new cut point for depression score of 9 was found to correspond to the original cut point score of 16 in the full CES-D scale.

CONCLUSIONS

This Rasch analysis of the CES-D in a longstanding RA cohort resulted in the construction of a modified 13-item scale with good internal validity. Further validation of the modified scale is recommended particularly in relation to the new cut point for depression.

The DIFs are a family of secreted chlorinated molecules that control cell fate during development of Dictyostelium cells in culture and probably during normal development too. They induce stalk cell differentiation and suppress spore cell formation. The biosynthetic and inactivation pathways of DIF-1 (the major bioactivity) have been worked out. DIF-1 is probably synthesised in prespore cells and inactivated in prestalk cells, by dechlorination. Thus, each cell type tends to alter DIF-1 level so as to favour differentiation of the other cell type. This relationship leads to a model for cell-type proportioning during normal development.

BACKGROUND

The Edinburgh Postnatal Depression Scale (EPDS) is a 10 item self-rating post-natal depression scale which has seen widespread use in epidemiological and clinical studies. Concern has been raised over the validity of the EPDS as a single summed scale, with suggestions that it measures two separate aspects, one of depressive feelings, the other of anxiety.

METHODS

As part of a larger cross-sectional study conducted in Melbourne, Australia, a community sample (324 women, ranging in age from 18 to 44 years: mean = 32 yrs, SD = 4.6), was obtained by inviting primiparous women to participate voluntarily in this study. Data from the EPDS were fitted to the Rasch measurement model and tested for appropriate category ordering, for item bias through Differential Item Functioning (DIF) analysis, and for unidimensionality through tests of the assumption of local independence.

RESULTS

Rasch analysis of the data from the ten item scale initially demonstrated a lack of fit to the model with a significant Item-Trait Interaction total chi-square (chi Square = 82.8, df = 40; p < .001). Removal of two items (items 7 and 8) resulted in a non-significant Item-Trait Interaction total chi-square with a residual mean value for items of -0.467 with a standard deviation of 0.850, showing fit to the model. No DIF existed in the final 8-item scale (EPDS-8) and all items showed fit to model expectations. Principal Components Analysis of the residuals supported the local independence assumption, and unidimensionality of the revised EPDS-8 scale. Revised cut points were identified for EPDS-8 to maintain the case identification of the original scale.

CONCLUSIONS

The results of this study suggest that EPDS, in its original 10 item form, is not a viable scale for the unidimensional measurement of depression. Rasch analysis suggests that a revised eight item version (EPDS-8) would provide a more psychometrically robust scale. The revised cut points of 7/8 and 9/10 for the EPDS-8 show high levels of agreement with the original case identification for the EPDS-10.

The differentiation-inducing signals (DIFs) currently known in Dictyostelium appear unable to account for the full diversity of cell types produced in development. To search for new signals, we analyzed the differentiation in monolayers of cells expressing prestalk (ecmAO, ecmA, ecmO, ecmB and cAR2) and prespore (psA) markers. Expression of each marker drops off as the cell density is reduced, suggesting that cell interaction is required. Expression of each marker is inhibited by cerulenin, an inhibitor of polyketide synthesis, and can be restored by conditioned medium. However, the known stalk-inducing polyketide, DIF-1, could not replace conditioned medium and induce the ecmA or cAR2 prestalk markers, suggesting that they require different polyketide inducers. Polyketide production by fungi is stimulated by cadmium ions, which also dramatically stimulates differentiation in Dictyostelium cell cultures and the accumulation of medium factors. Factors produced with cadmium present were extracted from conditioned medium and fractionated by HPLC. A new factor inducing prespore cell differentiation, called PSI-2, and two inducing stalk cell differentiation (DIFs 6 and 7) were resolved. All are distinct from currently identified factors. DIF-6, but not DIF-7 or PSI-2, appears to have an essential carbonyl group. Thus Dictyostelium may use extensive polyketide signaling in its development.

OBJECTIVE

The aim of this study was to adapt the Rheumatoid Arthritis Quality of Life (RAQoL) questionnaire for use in Turkey and to test its reliability and validity.

METHODS

The translation process included the recent guidelines for cross-cultural adaptation. Reliability of the Turkish RAQoL was assessed by internal consistency and test-retest reliability, internal construct validity by Rasch analysis, and external construct validity by associations with impairments, disability, and general health status. Cross-cultural validity was tested through analysis of differential item functioning (DIF) by comparison with data from the UK version of the RAQoL.

RESULTS

Reliability of the adapted version was good, with high internal consistency (Cronbach's alpha 0.95 and 0.96 at times 1 and 2, respectively) and test-retest reliability (Spearman's rho 0.874). Internal construct validity was confirmed by excellent fit to the Rasch model (mean item fit 0.236, SD 1.113) and external construct validity by expected associations. The DIF for culture was found in four items.

CONCLUSIONS

Adaptation of the RAQoL for use in Turkey was successful. The instrument can be used in both national and international studies for cross-cultural comparison with the UK, as long as adjustments are made for the few items displaying DIF for culture.

This study evaluated and compared the measurement properties of the 13-item Functional Assessment of Chronic Illness Therapy-Fatigue Scale (FACIT-F) and the 9-item Fatigue Severity Scale (FSS) in 118 consecutive Parkinson's disease (PD) patients, using traditional and Rasch measurement methodologies. Both questionnaires exhibited excellent data quality and reliability (coefficient alpha>or=0.9), and acceptable rating scale functionality, and both discriminated between fatigued and nonfatigued patients. Factor and Rasch analyses provided general support for unidimensionality of both FACIT-F and FSS, although they do not appear to measure identical aspects of fatigue. No signs of differential item functioning (DIF) were found for the FACIT-F, whereas potential age DIF was detected for two FSS items. These results support the measurement validity of both questionnaires in PD, although the FACIT-F displayed better measurement precision and modest psychometric advantages over the FSS. Availability of psychometrically sound fatigue measures that are applicable across disorders provides a sound basis for advancing the understanding of this common and distressing complaint.

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.

BACKGROUND

Dermatitis herpetiformis (DH) is a cutaneous manifestation of gluten sensitivity, occasionally associated with other autoimmune disorders, and reportedly associated with an increased risk of lymphoproliferative disorders. We describe a series of patients with DH, focusing on associated disorders (particularly celiac disease), incidence of lymphoma, histopathology, and sensitivity of direct immunofluorescence (DIF) testing and serologic testing with antiendomysium antibodies for the diagnosis of DH.

METHODS

The medical records of 264 patients with DH diagnosed between 1970 and 1996 were reviewed retrospectively. In addition, the records of six patients evaluated before the advent of DIF testing between 1932 and 1969 were reviewed.

RESULTS

Established celiac disease was present in 12.6% of patients with DH, autoimmune systemic disorders in 22.2%, malignant neoplasms in 10.4%, sarcoidosis in four patients, and ulcerative colitis in six patients. Lymphoproliferative disorders were found in seven patients. The histopathologic examinations showed a marked predominance of neutrophils in the inflammatory infiltrate. DIF testing was positive in 92.4% of the patients tested. Indirect immunofluorescence assay indicated circulating antiendomysial antibodies in the sera of 40 of the 63 patients tested (63.5%).

CONCLUSIONS

In this large series of patients with DH from a single institution, patients had a low incidence of symptomatic gluten-sensitive enteropathy, low risk of lymphoproliferative disorders, and associations with other systemic autoimmune disorders. The value of DIF testing in the diagnosis of DH was confirmed. The detection of antiendomysial antibodies by indirect immunofluorescence was less sensitive than indicated by other reports.

Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba.

Neurodegeneration is a hallmark of the human disease ataxia-telangiectasia (A-T) that is caused by mutation of the A-T mutated (ATM) gene. We have analyzed Drosophila melanogaster ATM mutants to determine the molecular mechanisms underlying neurodegeneration in A-T. Previously, we found that ATM mutants upregulate the expression of innate immune response (IIR) genes and undergo neurodegeneration in the central nervous system. Here, we present evidence that activation of the IIR is a cause of neurodegeneration in ATM mutants. Three lines of evidence indicate that ATM mutations cause neurodegeneration by activating the Nuclear Factor-κB (NF-κB) transcription factor Relish, a key regulator of the Immune deficiency (Imd) IIR signaling pathway. First, the level of upregulation of IIR genes, including Relish target genes, was directly correlated with the level of neurodegeneration in ATM mutants. Second, Relish mutations inhibited upregulation of IIR genes and neurodegeneration in ATM mutants. Third, overexpression of constitutively active Relish in glial cells activated the IIR and caused neurodegeneration. In contrast, we found that Imd and Dif mutations did not affect neurodegeneration in ATM mutants. Imd encodes an activator of Relish in the response to gram-negative bacteria, and Dif encodes an immune responsive NF-κB transcription factor in the Toll signaling pathway. These data indicate that the signal that causes neurodegeneration in ATM mutants activates a specific NF-κB protein and does so through an unknown activator. In summary, these findings suggest that neurodegeneration in human A-T is caused by activation of a specific NF-κB protein in glial cells.

The individual amoebae composing of Dictyostelium aggregate can differentiate into either stalk or spore cells according, it is believed, to the extracellular signals they receive. By inducing the differentiation of isolated cells we hope to identify these signals. It is shown here that wild-type cells can differentiate into prespore cells in a solution of cAMP plus salts supplemented by conditioned medium. Cell-to-cell contact is not required. More important, isolated cells of various sporogenous mutant strains form spores in similar conditions without needing conditioned medium at all. For these strains at least, cAMP is the sole inducer necessary for spore formation. Earlier work has shown that stalk cells are induced by a combination of cAMP and a low molecular weight factor, differentiation inducing factor (DIF). DIF now appears to be the only pathway-specific inducer involved in the differentiation of sporogenous amoebae and DIF levels in the aggregate may therefore determine whether an amoeba becomes a stalk or a spore cell. In suitable conditions some sporogenous mutants form migrating slugs having an anterior/posterior pattern of prestalk and prespore cells. This pattern could be generated by the localized activity of DIF.

Two endogenous differentiation-inducing factors (DIF-2 and DIF-3), which induce stalk-cell differentiation in the cellular slime mould Dictyostelium discoideum, have been identified as the pentan-1-one and monochloro analogues respectively of (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one). These compounds represent a new chemical class of effector molecules.