OBJECTIVE

We examined the presence and impact of differential item functioning (DIF) in a set of knee-specific functional status (FS) items administered using computerized adaptive testing (CAT) among English (United States) and Hebrew (Israel) speaking patients receiving therapy for knee impairments. DIF occurs in an item if probabilities of endorsing responses differ across groups after controlling for the FS measured.

METHODS

We analyzed data from 28,320 patients (14,160 U.S., 14,160 Israel) who completed the knee-specific CAT. Items were assessed for DIF by gender, age, symptom acuity, surgical history, exercise history, and language spoken using a hybrid technique that combines multiple ordinal logistic regression and item response theory FS estimates.

RESULTS

Several items had non-uniform DIF for covariates including language, but unadjusted and DIF-adjusted functional status estimates were in strong concordance [ICC(2,1) values>>/=0.97], and differences between unadjusted and adjusted FS scores represented <0.4% of the unadjusted FS standard deviation.

CONCLUSIONS

Statistically significant DIF was identified in some items but represented negligible clinical impact. Results suggested no need to adjust items for DIF when assessing FS outcomes across groups of patients with knee impairments who answer the knee CAT items in English in the United States or Hebrew in Israel. These findings suggest negligible differences in cultural perceptions between English and Hebrew wording of these knee-specific CAT FS items.

p22/PACAP response gene-1 (PRG1) is a novel rat early response gene expressed during induction of proliferation and stress response. In humans, a homolog of p22/PRG1, designated IEX-1/DIF-2, exists, yet the exact function of this gene remains elusive. To characterize the expression of p22/PRG1 in human cancers, we analyzed the expression of p22/PRG1 in the human pancreatic carcinoma cell lines 818-4, PT45, and PancTu1. Serum or growth factors, like epidermal growth factor (EGF) and hepatocyte growth factor (HGF), rapidly and transiently induced transcription of p22/PRG1 in these cells, correlating with the mitogenic response. Treatment with TNF-alpha was followed by a rapid increase of p22/PRG1 messenger RNA (mRNA) levels in PT45 and Panc-Tul cells, which proliferate in the presence of TNF-alpha, but not in 818-4 cells, which are growth-inhibited when treated with TNF-alpha. Our findings suggest that human p22/PRG1 is expressed in various pancreatic carcinoma cells as a growth-associated early response gene.

Xenorhabdus nematophila/Steinernema carpocapsae and Photorhabdus luminescens/Heterorhabditis bacteriophora are nemato-bacterial complexes highly pathogenic for insects. Using a syringe as artificial vector, we have analyzed the effects of the two bacteria, X. nematophila and P. luminescens on the genetic tool insect, Drosophila melanogaster. Both bacteria were found to kill adult flies in a dose dependent manner with X. nematophila being the fastest. On the other hand, when an injection of non-pathogenic bacteria, Escherichia coli, is performed 1 day before challenge with the entomopathogenic bacteria, then the survival of Drosophila flies was prolonged by at least 20h. After injection of entomopathogenic bacteria, Drosophila mutant Dif(1), affected on the Toll pathway, showed a similar phenotype than wild-type flies whereas Drosophila mutant Dredd(D55), affected on the imd pathway, was not protected by a prior injection of E. coli. This suggested that members of the imd pathway might be targets of these entomopathogenic bacteria albeit synthesis of antimicrobial peptides through this signaling pathway was induced by X. nematophila as well as P. luminescens. Finally, P. luminescens phoP mutant, an avirulent mutant in the Lepidopteran insect, Spodoptera littoralis, was found poorly virulent for D. melanogaster. phoP mutant partially protected D. melanogaster flies if injected 1 day before the injection of P. luminescens wild-type TT01 to the same extent than the E. coli-induced protection. However, phoP recovered a level of pathogenicity comparable to P. luminescens wild-type TT01 when injected to Drosophila flies affected on the imd pathway.

BACKGROUND

There is a growing interest in new therapeutic strategies for the treatment of Alzheimer disease (AD) which focus on reducing the beta-amyloid peptide (Aβ) burden in the brain by sequestering plasma Aβ, a large proportion of which is bound to albumin and other proteins. This review discusses the concepts of interaction between Aβ and albumin that have given rise to AMBAR (Alzheimer's Disease Management by Albumin Replacement) project, a new multicentre, randomised, controlled clinical trial for the treatment of AD.

METHODS

Results from preliminary research suggest that Albutein(®) (therapeutic albumin, Grifols) contains no quantifiable levels of Aβ. Studies also show that Albutein(®) has Aβ binding capacity. On the other hand, AD entails a high level of nitro-oxidative stress associated with fibrillar aggregates of Aβ that can induce albumin modification, thus affecting its biological functions. Results from the phase ii study confirm that using therapeutic apheresis to replace endogenous albumin with Albutein(®) 5% is feasible and safe in patients with AD. This process resulted in mobilisation of Aβ and cognitive improvement in treated patients. The AMBAR study will test combination therapy with therapeutic apheresis and haemopheresis with the possible leverage effect of Albutein(®) with intravenous immunoglobulin replacement (Flebogamma(®) DIF). Cognitive, functional, and behavioural changes in patients with mild to moderate AD will be assessed.

CONCLUSIONS

the AMBAR study represents a new therapeutic perspective for AD.

OBJECTIVE

To examine whether the personality dimensions, neuroticism and alexithymia, and the affective state dimensions measuring negative and positive affect significantly contributed to changes over time in the number of medically unexplained symptoms (MUS) reported.

METHODS

A total of 318 patients, presenting to their primary care physician with MUS, participated in the study. Logistic regression analyses were conducted to assess to what extent neuroticism, alexithymia, negative affect and positive affect independently contributed to (1) increase vs. decrease in the number of symptoms reported, and (2) the presence of a consistently high number of MUS over a 6-month follow-up period.

RESULTS

Negative affect was the strongest determinant of changes in the number of symptoms reported. In addition, low positive affect significantly contributed to changes in the number of symptoms over time. Next to negative affect, the dimension of alexithymia measuring difficulty in identifying feelings (DIF) was found to be an independent predictor of a consistently high number of MUS. Neither neuroticism nor general alexithymia independently contributed to changes in the number of symptoms over time or to symptom persistence.

CONCLUSIONS

Negative affect is an important determinant of MUS, because it contributes both to symptom evolution and symptom persistence. Positive affect has a beneficial effect on somatic symptom evolution, whereas the alexithymia dimension measuring DIF clearly contributes to the prediction of symptom persistence.

The treatment of primary breast cancer usually consists of surgery often followed by adjuvant therapy (radiotherapy, chemotherapy, hormonal treatment, etc.) to reduce the risk of recurrence. The cancer diagnosis and the treatments may have significant impact on the patients' quality of life. This thesis deals with scientific aspects and clinical results of a study aimed at assessing the impact of breast cancer (and its treatment) on the patients' quality of life. Studies such as this assessing the problems and symptoms experienced by the patients are often referred to as health-related quality of life (HRQL) research. HRQL research deals with subjective experiences and raises challenging, scientific questions. Therefore, much attention was directed towards methodological issues in this clinically motivated project. The study was a prospective, longitudinal, questionnaire-based investigation of women with newly diagnosed breast cancer registered in the Danish Breast Cancer Co-operative Group's DBCG 89 Program. The patients were sub-divided into low-risk and high-risk patients. High-risk patients were offered randomisation in one of three randomised adjuvant therapy trials involving chemotherapy, ovarian ablation, and endocrine therapy. After a literature study and interviews with breast cancer patients, a questionnaire was composed that included two widely used standard questionnaires (EORTC QLQ-C30 and Hospital Anxiety and Depression (HAD) Scale) and a DBCG 89 Questionnaire developed for this study. A total of 1,898 eligible patients were invited by post to participate in the study involving six assessments over a 2-year period, and 1,713 patients (90%) completed the first questionnaire. Furthermore, a questionnaire was sent to 872 women selected at random from the general population; 608 (70%) responded. The multi-item scales of the two standard questionnaires were analysed for so-called differential item functioning (DIF) in order to investigate whether the (summary) scale scores were adequate representations of the information obtained by the individual items. The DIF analyses identified a number of cases of DIF, which, among other things, contributed to detection of possible problems in the HAD Scale. It was concluded that DIF analyses are relevant when important analyses based on multi-item scales are made. A new way to evaluate the validity of questionnaires was developed. The results from questionnaires completed by patients were compared against results from open ended interviews with the same patients rated by observers. The idea was that if results were similar, the patients had then probably understood and completed the questionnaire items as intended. On the other hand, if results from self-assessment and interviews deviated, misunderstandings or other errors might have taken place, and the study would give insight into possible problems. Of 57 breast cancer patients, 46 (81%) were successfully interviewed. In general, the agreement between patient-completed questionnaires and interviews was excellent, indicating very good validity. The median weighted kappa for the EORTC QLQ-C30 was 0.85 (range 0.49-1.00); it was 0.79 (range 0.65-0.95) for the HAD Scale, and 0.92 (range 0.51-1.00) for the DBCG 89 Questionnaire. However, the study identified a mechanism called selective reporting, which may affect results from most HRQL questionnaires: in order to provide correct and useful answers some patients do not report symptoms they believe are irrelevant to the study, e.g., symptoms unrelated to cancer. This mechanism may lead to bias if results from patients are compared to results from populations reporting their symptoms more completely, e.g., general population samples. In contrast, this mechanism has little importance when results from different sub-groups of cancer patients are compared. In this study multiple variables were assessed at multiple points in time and we did not have a priori hypotheses for all these potential comparisons. Therefore, a staff survey involving experienced doctors and nurses was conducted in order to generate hypotheses that could be tested in the data from patients. We contacted 46 health care professionals and 36 (78%) responded. Overall, the staff survey did not prove very useful for the intended purpose. The main reason for this was probably that the health care professionals had limited insight into the patients' HRQL. A different approach to the problem of multiple hypothesis testing proved more useful. Hypotheses generated from the initial literature review were tested in the comparison of patients in chemotherapy against patients not in chemotherapy. The study of women selected at random from the general population showed that these women experienced a considerable degree of "morbidity" according to all three questionnaires. This shows that symptoms and problems reported by cancer patients may have causes other than cancer, and thus constitutes a good justification for the use of data from general population studies when interpreting data from cancer patients. The levels of anxiety and depression of low-risk breast cancer patients were found to be lower than those from the general population sample. After careful consideration we concluded that this finding was probably incorrect. The most important explanations were thought to be the wording of some HAD Scale items as well as two mechanisms that are not specific to the HAD Scale, the "selective reporting mechanism" found in the validation study, and the response-shift problem. These findings indicate - in contrast to the conclusion above - that the comparability of HRQL data from cancer patients and general population data must be questioned. However, as this is the first study to raise the problem, this issue needs further investigation. Based on the initial literature review and interviews we hypothesised that 30 different HRQL issues would be impaired in patients undergoing CMF chemotherapy compared to patients not in chemotherapy; 23 of these hypotheses were confirmed. In addition, our study and other research suggest that other HRQL aspects may also be affected by chemotherapy. Thus, there is considerable evidence that patients in chemotherapy may experience effects on a wide spectrum of HRQL issues. Most other studies have assessed surprisingly few of the HRQL issues shown in our study to be impaired in patients receiving chemotherapy. Similarly, current review articles on HRQL effects of adjuvant chemotherapy mention only relatively few of these topics. Concerning HRQL after the treatment period, our main finding was that many symptoms and problems had declined or disappeared, but some persisted: anticipatory nausea, weight gain, endocrine effects (e.g., hot flushes/sweats, irregular bleedings/amenorrhea, vaginal dryness), disturbed sleep, and sexual dysfunction. These findings are in agreement with the literature. The staff study showed that experienced physicians and nurses did not expect many of the "scientifically well documented" consequences of chemotherapy. Taken together, our findings suggest that information to patients about chemotherapy should be more comprehensive than that which has been practised in most places. When compared against ovarian ablation, chemotherapy was associated with more impact on HRQL during the treatment period; only hot flushes/sweats were more pronounced in the ovarian ablation group. Thus, from an overall "HRQL perspective" ovarian ablation or suppression may be preferable. However, younger women may preserve their premenopausal status (including fertility) by having chemotherapy, and this may be an argument for chemotherapy or for temporary ovarian ablation via goserelin, rather than permanent ovarian ablation. Furthermore, while ovarian ablation/suppression may be preferable because of less impairment of HRQL, contemporary chemotherapeutic regimens may be more effective. These results indicate that for some patients, the HRQL data and results on treatment efficiency may be in conflict. There is no simple, universally correct solution to this dilemma. More research into patients' views and expectations to the health-care system in cases where medical decision-making involves complex trade-offs between treatment efficiency and HRQL issues is needed. Contrary to expectations, the analyses showed that fatigue and emotional function predicted the risk of recurrence and death independently of biological and clinical prognostic variables. In multivariate Cox regression analyses patients who were more fatigued or had poorer emotional function had a worse prognosis. These results are consistent with one small study, but are inconsistent with five similar studies in patients with primary breast cancer, which found no such associations. The reasons for these important differences are currently unknown. In conclusion, this study consisted of methodological and clinical investigations of HRQL in primary breast cancer patients. The initial questionnaire development resulted in a combination of questionnaires that was more comprehensive than in other similar studies. The results of the methodological studies generally supported the validity of the questionnaires but also gave important insights into potential scientific problems that are probably not restricted to the present study. These insights helped to prevent misinterpretations of the clinical data. The study provided the most detailed description of HRQL during and after breast cancer adjuvant chemotherapy to date, and compared results of chemotherapy against ovarian ablation. It also provided controversial results concerning the prognostic value of HRQL data. The combination of a large empirical study and several methodological sub-studies thus proved useful and gave new results.

Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2-dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30-amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type-specific, these studies suggest that breast cancer cells contain a novel "death switch" that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis.

The pDd63 and pDd56 genes encode extracellular matrix proteins which, respectively, surround the migratory slug and mature stalk cells. Both genes are dependent for their expression upon, and rapidly induced by, DIF, the stalk cell inducer. Using these genes as cell-autonomous markers, we have defined three distinct kinds of 'prestalk' cells localized to different parts of the anterior region of the slug. At least one, and probably both, prestalk cell types initially differentiates at the base of the aggregate. The most abundant of the two prestalk cell types then migrates into the tip, the precursor of the prestalk zone which arises at the apex of the aggregate. Thus we believe that morphogenesis of the prestalk zone, the primary pattern-forming event in Dictyostelium development, involves a combination of positionally localized differentiation and directed cell migration. To account for the positionally localized differentiation of prestalk cells, we invoke the existence of gradients of the known antagonists of DIF-cAMP and NH3. We further suggest that differences in the motility of pstA and pstB cells might result from differences in their chemotactic responsiveness to cAMP signals propagated from the tip.

OBJECTIVE

We assessed the measurement equivalence and feasibility of the paper-and-pencil and touch-screen modes of administration of the Taiwan Chinese version of the EORTC QLQ-PR25, a commonly used questionnaire to evaluate the health-related quality of life (HRQOL) in patients with prostate cancer in Taiwan.

METHODS

A cross-over design study was conducted in 99 prostate cancer patients at an urology outpatient clinic. Descriptive exact and global agreement percentages, intraclass correlation, and equivalence test based on minimal clinically important difference (MCID) approach were used to examine the equity of HRQOL scores between these two modes of administration. We also evaluated the feasibility of computerized assessment based on patients' acceptability and preference. Additionally, we used Rasch rating scale model to assess differential item functioning (DIF) between the two modes of administration.

RESULTS

The percentages of global agreement in all domains were greater than 85% in the EORTC QLQ-PR25. All results from equivalence tests were significant, except for Sexual functioning, indicating good equivalence. Only one item exhibited DIF between the two modes. Although nearly 80% of the study patients had no prior computer-use experience, the overall proportion of acceptance and preference for the touch-screen mode were quite high and there was no significant difference across age groups or between computer-use experience groups.

CONCLUSIONS

The study results showed that the data obtained from the modes of administration were equivalent. The touch-screen mode of administration can be a feasible and suitable alternative to the paper-and-pencil mode for assessment of patient-reported outcomes in patients with prostate cancer.

The purpose of this study was to determine if the Mini-Mental State Examination (MMS; M. F. Folstein, S. E. Folstein, & P. R. McHugh, 1975) demonstrates item bias with respect to measuring cognitive functioning of older Hispanics and non-Hispanics. Assessment of differential item functioning (DIF) of individual MMS items across 3 language/ethnicity groups (English test administration/non-Hispanic ethnicity, English test administration/Hispanic ethnicity, and Spanish test administration/Hispanic ethnicity) was performed by using a logistic regression procedure. Fifteen of the 26 MMS items were significantly related to total score and were shown to provide unbiased measurement across the 3 groups. Normative data are presented for older Hispanics (n = 365) and non-Hispanics (n = 388) on the raw MMS, a 15-item version in which items with significant DIF were eliminated, and a total score statistically adjusted for effects of education and age.

The migratory slug of Dictyostelium discoideum is surrounded by, and continuously synthesizes, an extracellular protein-cellulose matrix known as the slime sheath which is deposited on the substratum as a trail marking the slug's progress. We show that the stalk-specific proteins, ST310 and ST430, are exclusively located in the slime sheath and trail and that fusion genes, containing upstream sequences from the cognate genes, direct correct mRNA accumulation during development and correct localization of the fusion protein. Immunoelectron microscopy shows the ST310 and ST430 proteins to be present throughout the entire thickness of the slime sheath and almost totally absent from the cells of the slug. The genes that encode the ST310 and ST430 polypeptides are inducible by DIF, a stalk-specific inducing agent, and the mRNAs are highly enriched in prestalk over prespore cells. The production of these extracellular proteins by prestalk cells suggests that, in a manner somewhat analogous to that of extracellular matrix proteins of higher eukaryotes, the anterior region of the slug may be responsible for the continuous deposition of a track, along which the slug cells migrate. In the mature culminant, the ST310, and possibly the ST430, protein form part of the stalk tube and stalk cell wall. Therefore, the results also show that there are proteins common to both slime trial and stalk tube, indicating a possible precursor-product relationship between these chemically similar integuments.

Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of G(salpha), causing resistance to parathyroid hormone and other hormones that transduce signals via G(s). Most G(salpha)mutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant G(salpha) (alphas-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that alphas-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. alphas-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) alphas-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in alphas palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, alphas-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of alphas-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of G(salpha) in a cell-specific manner.

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is β-arrestin Kurtz (Krz), which we previously implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. In this study, we have uncovered a link between these two modes of regulation and show that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, we found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. Our results demonstrate that β-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation.

Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin-Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

From the sequences of Rel/NF-kappa B and I kappa B proteins, we constructed an alignment of their Rel Homology Domain (RHD) and ankyrin repeat domain. Using this alignment, we performed tree reconstruction with both distance matrix and parsimony analysis and estimated the branching robustness using bootstrap resampling methods. We defined four subfamilies of Rel/NF-kappa B transcription factors: (i) cRel, RelA, RelB, Dorsal and Dif; (ii) NF-kappa B1 and NF-kappa B2; (iii) Relish and (iv) NF-AT factors, the most divergent members. Subfamilies I and II are clustered together whereas Relish diverged earlier than other Rel/NF-kappa B proteins. Three subfamilies of I kappa B inhibitors were also defined: (i) NF-kappa B1 and NF-kappa B2; (ii) close to subfamily I, the short I kappa B proteins I kappa B alpha, I kappa B beta and Bcl-3; (iii) Relish that diverged earlier than other I kappa B inhibitors. Our definition of groups and subfamilies fits to structural and functional features of the Rel/NF-kappa B and I kappa B proteins. We also showed that ankyrin repeats of NF-kappa B1, NF-kappa B2 and Relish are short I kappa B-specific ankyrin motifs. These proteins defining a link between Rel/NF-kappa B and I kappa B families, we propose that all these factors evolved from a common ancestral RHD-ankyrin structure within a unique superfamily, explaining the specificities of interaction between the different Rel/NF-kappa B dimers and the various I kappa B inhibitors.

DIF-1 [Differentiation-Inducing Factor 1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one] is a novel chlorinated signal molecule that induces stalk-cell differentiation during development of Dictyostelium discoideum. Here we introduce the use of the radioisotope 36Cl to label DIF-1 and other low-Mr chlorinated compounds produced during development. H.p.l.c. and t.l.c. were used to resolve the labelled compounds. We find the following. (1) At least 14 dialysable 36Cl-labelled compounds are released into the medium by cells labelled continuously through development with Na36Cl. (2) The compounds can be classified into two major groups according to their times of accumulation in development. The early group of compounds starts accumulating at the end of aggregation, co-ordinately with DIF-1; the late group is only made at the end of development, by mature fruiting bodies. There may also be an intermediate group made during culmination. (3) The early group of compounds has been identified as comprising DIF-1 and seven of its metabolites by co-chromatography with the authentic compounds. These metabolites had previously only been recognized in suspensions of living cells incubated with exogenous DIF-1. Their detection here, from cells undergoing normal development, suggests that endogenous DIF-1 is metabolized in normal development in much the same way as is DIF-1 added to cells in suspension. (4) The intermediate and late groups of compounds are not obvious DIF-1 metabolites. They may have some role unconnected with DIF signalling. (5) A group of 36Cl-labelled late compounds remain cell-associated after washing of the fruiting bodies, and these are greatly enriched in stalk, compared with spore, cells. (6) Other slime-mould species were labelled with 36Cl. All three tested, namely D. mucoroides, D. vinaceo-fuscum and P. violaceum, also produced chloro compounds. D. mucoroides produced DIF-1 by the criterion of h.p.l.c. co-elution with authentic DIF-1. A developmentally regulated metabolism of chlorinated compounds may therefore be widespread amongst slime moulds. To our knowledge, labelling with 36Cl in vivo has not been reported before and provides a powerful general method for investigating chlorinated compounds in diverse organisms.

BACKGROUND

Previous studies have demonstrated that several N-substituted 4', 4″-diF-benztropine (BZT) analogs with high dopamine transporter affinity selectively decreased cocaine self-administration without affecting food-maintained behavior in rats.

OBJECTIVE

The present study examined if the decreases in cocaine self-administration are due to competition from excess behavioral activity (hyperlocomotion or stereotypy) induced by the BZT analogs alone or in combination with cocaine.

RESULTS

Pretreatments with the typical dopamine uptake inhibitor methylphenidate [1.0, 3.2, and 10 mg/kg, intraperitoneally (i.p.)] dose-dependently shifted the cocaine self-administration dose-effect curve (0, 0.032, 0.1, 0.32, and 1.0 mg/kg/injection) leftward. The shift in the dose-effect curve was obtained at doses of methylphenidate that, when administered alone, also decreased food-maintained behavior and increased locomotor activity and stereotypy. In contrast, the N-substituted BZT analogs, JHW 007 (1.0, 3.2, and 10 mg/kg, i.p.), AHN 1-055 (10 mg/kg), and, AHN 2-005 (10 mg/kg), as previously reported, decreased the maximum for the cocaine self-administration dose-effect curve, and did so at doses that were virtually without effects on food-maintained behavior. Further, the BZT analogs alone had minimal effects on locomotor activity and stereotypies and did not appreciably change the effects of cocaine on these measures when administered in combination with cocaine.

CONCLUSIONS

The present results suggest that the decrease in cocaine self-administration produced by the N-substituted BZT analogs is due to an antagonism of the reinforcing effects of cocaine rather than due to interference from competing behavioral overstimulation, and further supports the development of N-substituted BZT analogs as medications to treat cocaine abuse.

The Rel protein Dif is a transcription factor suggested to control part of the immune response in the fruit fly Drosophila melanogaster. In uninfected animals, Dif is normally located in the cytoplasm, most likely in a complex with an IkappaB molecule such as Cactus. Upon infection, Dif is enriched in the nucleus of immunoresponsive tissues such as fat body and blood cells. Rel proteins in mammals not only participate in the control of the immune response, but are also thought to play important roles in the function of the nervous system. Here, we demonstrate that both Dif and Cactus are expressed in the central nervous system (CNS) of Drosophila. Interestingly, Dif and Cactus colocalize in their distribution, suggesting a functional link between these proteins in the CNS. In the larval CNS, both Dif and Cactus are expressed at relatively low levels in most cells and at high levels in the mushroom bodies and in small subsets of neurosecretory cells. The cytoplasmic localization of Dif and Cactus in the CNS cells is not affected by bacterial challenge. Instead, we observed changes in nuclear versus cytoplasmic localization of Cactus (but not Dif) along the dark-light cycle, with a strong nuclear localization in perineurial glia toward the end of the dark period. In the CNS of the prepupa, the intensity of the immunostaining for both Dif and Cactus is higher than in the larva. Interestingly, in fat body of uninfected prepupae, the Dif localization was mainly nuclear, suggesting a function for Dif during the process of pupariation.

In search for alternative methods for developmental toxicity testing, we investigated whether embryonic stem cell (ESC) differentiation and its modulation by toxic exposure could be monitored by proteome profiling. We compared the proteomes of mouse ESC, differentiating ESC (DIF) and differentiating ESC exposed to the embryotoxicant monobutyl phthalate (MBP). Experiments were performed in duplicates for each cell culture and the proteins extracted from the cells were separated by one-dimensional SDS-PAGE. The identified proteins were quantified using a label-free quantitative method based on counting the observed peptides as an index of protein abundance. Fifty-seven proteins were upregulated in DIF relative to ESC, whereas 42 proteins were downregulated. Most of the upregulated proteins could be correlated with cardiomyocyte functionality. In contrast, the downregulated proteins were principally pluripotency markers, chaperones and ribosomal proteins. Higher expression levels of enzymes involved in DNA mismatch repair (MSH6) and in methylation reactions (MAT2A, AHCY) were also detected in the ESC, suggesting that these processes are more active in the ESC. In addition, the detection of gluthatione S-transferase alpha 4 (GSTA4) and Park7 DJ-1 protein (antioxidant) in ESC indicates that these cells have potential detoxification mechanisms. Furthermore, MBP affected the expression of 33 proteins, including MYH6, a cardiomyocyte-specific protein, which was significantly downregulated. MBP also affected the expression levels of chaperones, metabolic enzymes and chromatin modulating proteins, suggesting that MBP alters the differentiation process. Western blot analysis of MYH6 and HSPB1 confirmed the proteomic data. In addition, a favorable correlation was observed between protein expression and published changes in the expression of related genes at the transcriptomics level. Together, the results reveal potential protein markers that may be used as endpoints in an ESC based animal free alternative test for embryotoxicity though further studies are required for confirmation.

The mode of action of difficidin (DIF) was investigated. Upon addition of DIF to log phase cultures of Escherichia coli, growth ceased immediately and small round cells accumulated after 30 minutes incubation. No cell lysis was observed. DIF was rapidly bactericidal to both growing and stationary phase cultures, and inhibited protein synthesis more rapidly than RNA, DNA, or cell-wall synthesis in growing cells. Protein synthesis was also inhibited in a cell-free system. The frequency of natural mutation to resistance in E. coli was less than 1 in 10(10) cells.

The choice of the stalk cell differentiation pathway in Dictyostelium is promoted by an endogenous substance, DIF-1, which is 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone. It is also favoured by weak acids and two inhibitors of the plasma membrane proton pumps of fungi and plants, diethylstilbestrol (DES) and zearalenone, and antagonised by ammonia and other weak bases, which promote spore differentiation. These observations led to the proposal that the choice of differentiation pathway is regulated by intracellular pH. They also prompted the conjecture that DIF-1 itself is a plasma membrane proton pump inhibitor. We report here experiments showing that DIF-1 is not a plasma membrane proton pump inhibitor. We demonstrate that diethylstilbestrol and zearalenone do inhibit the plasma membrane proton pump of Dictyostelium and we show that there is an excellent qualitative and quantitative correlation between the inhibitory activity of these agents, and of a number of other substances, and their ability to divert differentiation from the spore to the stalk pathway. We conclude that inhibition of the plasma membrane proton pump does shift the choice of differentiation pathway in Dictyostelium towards the stalk pathway, but that DIF does not act by this route, and we propose a model for the actions of DIF and plasma membrane proton pump inhibitors in which the differentiation pathway is controlled by the pH of intracellular vesicles rather than by intracellular pH itself. The model invokes a DIF- and proton-activated vesicular chloride channel whose opening permits acidification of the vesicles and lowers cytosolic Ca++ concentration.

Solid dispersions of diflunisal (DIF) with Eudragit RS100 (RS) and RL100 (RL) with different drug-to-polymer ratios were prepared by a solvent method (coevaporates) and were characterised in the solid state in comparison with the corresponding physical mixtures. The work was aimed at characterising the interactions occurring between DIF and RS or RL polymers, along with their influence on the in-vitro drug-dissolution pattern. The findings suggest that the drug did not change its crystalline form within the polymer network. Drug dispersion in the polymer matrix strongly influences its dissolution rate, which appears slower and more gradual while increasing the polymer ratios. Moreover, DIF is known to be a photosensitive compound, and its photoproduct has been found to be a toxic agent. This can be evidenced by testing red blood cell membranes for their resistance to the osmotic shock induced by UVA irradiation in the presence of DIF. The presence of some DIF/RS coevaporates was shown to reduce significantly the drug photosensitization process towards cell membranes. This suggests the possibility of combining the design of a drug delivery system with a photoprotective strategy.

A remarkable property of some DNA-binding proteins that can interact with and pair distant DNA segments is that they mediate their biological function only when their binding sites are arranged in a specific configuration. Xer site-specific recombination at natural plasmid recombination sites (e.g., cer in ColE1) is preferentially intramolecular, converting dimers to monomers. In contrast, Xer recombination at the Escherichia coli chromosomal site dif can occur intermolecularly and intramolecularly. Recombination at both types of site requires the cooperative interactions of two related recombinases, XerC and XerD, with a 30-bp recombination core site. The dif core site is sufficient for recombination when XerC and XerD are present, whereas recombination at plasmid sites requires approximately 200 bp of adjacent accessory sequences and accessory proteins. These accessory factors ensure that recombination is intramolecular. Here we use a model system to show that selectivity for intramolecular recombination, and the consequent requirement for accessory factors, can arise by increasing the spacing between XerC- and XerD-binding sites from 6 to 8 bp. This reduces the affinity of the recombinases for the core site and changes the geometry of the recombinase/DNA complex. These changes are correlated with altered interactions of the recombinases with the core site and a reduced efficiency of XerC-mediated cleavage. We propose that the accessory sequences and proteins compensate for these changes and provide a nucleoprotein structure of fixed geometry that can only form and function effectively on circular molecules containing directly repeated sites.

In an effort to advance an understanding of the phenomenology of bipolar II depression, the current study used methods based in item response theory to evaluate differences in DSM-IV depression symptom endorsement in an epidemiological sample of individuals with a history of hypomania (i.e., bipolar II depression) in comparison to: a) individuals with a history of mania (i.e., bipolar I depression), and b) individuals without a history of hypomania or mania (i.e., unipolar depression). Clinical interview data were drawn from a subsample (n = 13,753) of individuals with bipolar II, bipolar I, or unipolar depression who had participated in the National Epidemiologic Survey on Alcohol and Related Conditions. A two-parameter item response model was used to estimate differential item functioning (DIF) between these groups. Differences in severity parameter estimates revealed that suicidal ideation/attempt was less likely to be endorsed across most levels of depression severity in bipolar II versus bipolar I disorder. There were no significant differences between groups on the remaining DSM-IV symptoms. Although preliminary, current study data are consistent with recent assertions that depression may be understood as a clinical phenomenon that is consistent across the major affective disorders. An exception to this conclusion may be in the area of suicidal ideation, which requires additional attention.