The independent role of prenatal and postnatal exposure to active and passive smoking on the development of early wheeze in children.

Maternal smoking during pregnancy increases childhood asthma risk, but health effects in children of nonsmoking mothers passively exposed to tobacco smoke during pregnancy are unclear. We examined the association of maternal passive smoking during pregnancy and wheeze in children aged ≤2 years.Individual data of 27 993 mother-child pairs from 15 European birth cohorts were combined in pooled analyses taking into consideration potential confounders.Children with maternal exposure to passive smoking during pregnancy and no other smoking exposure were more likely to develop wheeze up to the age of 2 years (OR 1.11, 95% CI 1.03-1.20) compared with unexposed children. Risk of wheeze was further increased by children's postnatal passive smoke exposure in addition to their mothers' passive exposure during pregnancy (OR 1.29, 95% CI 1.19-1.40) and highest in children with both sources of passive exposure and mothers who smoked actively during pregnancy (OR 1.73, 95% CI 1.59-1.88). Risk of wheeze associated with tobacco smoke exposure was higher in children with an allergic versus nonallergic family history.Maternal passive smoking exposure during pregnancy is an independent risk factor for wheeze in children up to the age of 2 years. Pregnant females should avoid active and passive exposure to tobacco smoke for the benefit of their children's health.

Screen time, adiposity and cardiometabolic markers: mediation by physical activity, not snacking, among 11-year-old children.

BACKGROUND: There is evidence for a relation of TV viewing with adiposity and increased cardiometabolic risk factors in children and adolescents. It is unclear to what extent this relation is mediated by snacking and lack of physical activity. We determined whether associations of screen time with adiposity and cardiometabolic markers were mediated by these behaviours. METHODS: Children from a population-representative Dutch birth cohort (n=1447) reported screen time and other lifestyle factors by a questionnaire around the age of 11 years (range 10-14) and had anthropometry and cardiometabolic markers measured around the age of 12 years (range 12-14). Adjusted associations of screen time with snacking, physical activity, adiposity and cardiometabolic markers (total-to-high-density lipoprotein cholesterol (TC/HDLC) ratio, blood pressure, glycated haemoglobin) were assessed by using formal mediation analysis. We tested the hypothesized paths by structural equation modeling, which allows quantification of the indirect effects associated with potential mediators. RESULTS: Children with ⩾20 h screen time per week consumed more snacks (1.9 vs 1.3 portions per day) and were less physically active (4.3 vs 4.8 days per week) than children with maximum 6 h screen time. Screen time was directly associated with higher adiposity (standardized ß=0.10-0.12 depending on the outcome, P<0.001), and indirectly through less physical activity. The association of screen time with TC/HDLC ratio was almost completely mediated by adiposity (ß=0.39, P<0.0001), and to a minor extent by physical activity (ß=-0.06, P=0.02). There was no direct association of screen time with TC/HDLC ratio. CONCLUSIONS: The adverse association of screen time with adiposity was partly mediated by physical activity, but not by snacking. The association of screen time with TC/HDLC ratio was almost completely mediated by adiposity. Our results may suggest that future efforts in society and public health should be directed to replace screen time with physical activity for reducing children's adiposity and cardiometabolic risk.

Perinatal risk factors for wheezing phenotypes in the first 8 years of life.

BACKGROUND: A novel data-driven approach was used to identify wheezing phenotypes in pre-schoolchildren aged 0-8 years, in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) birth cohort. Five phenotypes were identified: never/infrequent wheeze, transient early wheeze, intermediate onset wheeze, persistent wheeze and late onset wheeze. It is unknown which perinatal risk factors drive development of these phenotypes. OBJECTIVE: The objective of the study was to assess associations of perinatal factors with wheezing phenotypes and to identify possible targets for prevention. METHODS: In the PIAMA study (n = 3963), perinatal factors were collected at 3 months, and wheezing was assessed annually until the age of 8 years. Associations between perinatal risk factors and the five wheezing phenotypes were assessed using weighted multinomial logistic regression models. Odds ratios were adjusted for confounding variables and calculated with 'never/infrequent wheeze' as reference category. RESULTS: Complete data were available for 2728 children. Risk factors for transient early wheeze (n = 455) were male gender, maternal and paternal allergy, low maternal age, high maternal body mass index, short pregnancy duration, smoking during pregnancy, presence of older siblings and day-care attendance. Risk factors for persistent wheeze (n = 83) were male gender, maternal and paternal allergy, and not receiving breastfeeding for at least 12 weeks. Intermediate onset wheeze (n = 98) was associated with a lower birth weight and late onset wheeze (n = 45) with maternal allergy. CONCLUSION AND CLINICAL RELEVANCE: We identified different risk factors for specific childhood wheezing phenotypes. Some of these are modifiable, such as maternal age and body mass index, smoking, day-care attendance and breastfeeding, and may be important targets for prevention programmes.

Repeated measurements of mite and pet allergen levels in house dust over a time period of 8 years.

BACKGROUND: Studies of the association between indoor allergen exposure and the development of allergic diseases have often measured allergen exposure at one point in time. OBJECTIVE: We investigated the variability of house dust mite (Der p 1, Der f 1) and cat (Fel d 1) allergen in Dutch homes over a period of 8 years. METHODS: Data were obtained in the Dutch PIAMA birth cohort study. Dust from the child's mattress, the parents' mattress and the living room floor was collected at four points in time, when the child was 3 months, 4, 6 and 8 years old. Dust samples were analysed for Der p 1, Der f 1 and Fel d 1 by sandwich enzyme immuno assay. RESULTS: Mite allergen concentrations for the child's mattress, the parents' mattress and the living room floor were moderately correlated between time-points. Agreement was better for cat allergen. For Der p 1 and Der f 1 on the child's mattress, the within-home variance was close to or smaller than the between-home variance in most cases. For Fel d 1, the within-home variance was almost always smaller than the between-home variance. Results were similar for allergen levels expressed per gram of dust and allergen levels expressed per square metre of the sampled surface. Variance ratios were smaller when samples were taken at shorter time intervals than at longer time intervals. CONCLUSION: Over a period of 4 years, mite and cat allergens measured in house dust are sufficiently stable to use single measurements with confidence in epidemiological studies. The within-home variance was larger when samples were taken 8 years apart so that over such long periods, repetition of sampling is recommended.

Comparison of parental reports of smoking and residential air nicotine concentrations in children.

BACKGROUND: Using questionnaires to assess children's residential exposure to environmental tobacco smoke (ETS) may result in misclassification from recall and response bias. Questionnaire data have frequently been validated against urinary cotinine measurements, but rarely against actual measurements of residential air nicotine. OBJECTIVE: To compare questionnaire reported smoking with air nicotine concentrations in a large population of children and with urinary cotinine levels in a subpopulation; and to assess the potential impact of the symptom status of the children on the agreement between different measures of exposure. METHODS: The authors assessed residential exposure to ETS in 347 German, 335 Dutch, and 354 Swedish preschool and schoolchildren by questionnaire and air nicotine measurements, and in a subset of 307 German children by urinary cotinine measurements. They then compared the different measures of ETS exposure. RESULTS: In all countries, air nicotine concentrations increased with increasing questionnaire reported smoking in a dose-response fashion. Specificity and negative predictive values of questionnaire reports for nicotine concentrations were excellent. Sensitivity and positive predictive values were moderate to good. Excluding occasional smokers, the overall percentage of homes misclassified was 6.9%, 6.7%, and 5.1% in Germany, the Netherlands, and Sweden, respectively. Similar results were found for the agreement of urinary cotinine concentrations with questionnaire reports and air nicotine levels. There was no indication of underreporting by parents of symptomatic children. CONCLUSION: Despite some misclassification, questionnaire reports are an inexpensive and valid estimate of residential ETS exposure among preschool and school children.

Parental education and children's respiratory and allergic symptoms in the Pollution and the Young (PATY) study.

Inequalities in health between socio-economic groups are a major public health concern. The current authors studied associations between parental socio-economic status (SES) and children's respiratory and allergic symptoms in 13 diverse countries, including the Russian Federation, North America (Canada and the USA), and countries across Eastern and Western Europe. Data of 57,000 children aged 6-12 yrs, originating from eight cross-sectional studies, were analysed. SES was defined by parental education. Respiratory and allergic symptoms were defined by parental questionnaire reports. Multiple logistic regressions showed that low parental education was associated with a decreased risk of inhalant allergy and itchy rash in school children. Furthermore, low parental education was associated with an increased prevalence of wheeze and nocturnal dry cough. No clear association was found between parental education and prevalence of doctor-diagnosed asthma and bronchitis. Part of the difference between socio-economic groups with regard to their children's symptoms was explained by established risk factors, such as parental allergy, smoking during pregnancy, pet ownership, crowding, mould/moisture in the home, use of gas for cooking, and air pollution (particulate matter with a diameter of <10 microm). However, differences remained after adjusting for these variables. Children's health was associated with parental education. The association could not fully be explained by established risk factors.

Are house dust mite allergen levels influenced by cold winter weather?

BACKGROUND: Moisture is vitally important for house dust mites and they cannot survive in cold or hot-dry climates. AIMS OF THE STUDY: To investigate the influence of two extraordinarily cold and dry winters in 1995/1996 and 1996/1997 on house dust mite levels in German homes. METHODS: Dust samples were collected between June 1995 and December 2001 on the mattresses of 655 adults and 454 schoolchildren living in five different areas of Germany. We compared house dust mite allergen Dermatophagoides pteronyssinus (Der p 1) levels before and during the winters of 1995/1996 and 1996/1997 with levels after these winters. RESULTS: D. pteronyssinus (Der p 1) levels in samples taken after the cold winters of 1995/1996 and 1996/1997 were approximately two times lower than Der p 1 levels in dust samples collected before or during these respective winters (Geometric means: Erfurt 89 vs 33 ng/g; Hamburg 333 vs 219 ng/g; Bitterfeld, Hettstedt, and Zerbst 296 vs 180 ng/g). Except for Hamburg, the decrease in Der p 1 levels was statistically significant. D. pteronyssinus levels measured in dust samples collected in 2001 (i.e. 3 years after the two cold winters) show a statistically non-significant increase (Geometric means: Erfurt 33 vs 39 ng/g; Hamburg 219 vs 317 ng/g), suggesting that it may take a long time for mite allergen levels to increase again after a sudden decrease. CONCLUSION: We conclude that Der p 1 levels in German mattress dust samples have been approximately reduced by a factor of three to four by the two consecutive cold winters of 1995/1996 and 1996/1997.

The effect of storage on allergen and microbial agent levels in frozen house dust.

BACKGROUND: House dust samples collected for exposure studies are often stored for variable time periods until analysis. However, there is currently no information on the effects of dust storage on the content of biocontaminants. Therefore, associations were analysed between the levels of mite allergens (Der p 1, Der f 1), cat allergen (Fel d 1) and microbial components (endotoxin, beta(1-->3)-glucan) on the one hand and the storage duration of dust samples at -20 degrees C on the other hand. METHODS: Within the framework of a study on the influences of INdoor factors and Genetics on Asthma (INGA), dust samples were collected from living room floors between June 1995 and August 1998 and extracted according to a standardized protocol. The concentrations of Der p 1, Der f 1, Fel d 1 and beta(1-->3)-glucan were determined with specific enzyme immunoassays. Endotoxin content was quantified using a chromogenic kinetic Limulus amoebocyte lysate (LAL) test. All concentrations were expressed per gram of dust RESULTS: Dust samples (n = 1236) were obtained from 655 homes in Hamburg, Hettstedt, Zerbst and Bitterfeld. Storage duration (range 8-298 days) was grouped into four categories ( 120 d). After adjustment for city of residence and season of dust sampling, means ratios comparing categories 2-4 to the first category were not statistically significant for Der p 1, Der f 1, endotoxin and beta(1-->3 glucan). However, Fel d 1 concentrations significantly declined with increased storage times of dust samples. CONCLUSIONS: Storage of house dust at -20 degrees C for up to 10 months has no effect on mite allergen, endotoxin and beta(1-->3)-glucan levels. A potential loss of Fel d 1 during storage of frozen dust samples needs further investigations by repeated measurements of allergen in identical dust samples.

Maternal compliance with nutritional recommendations in an allergy preventive programme.

AIMS: To assess maternal compliance with nutritional recommendations in an allergy preventive programme, and identify factors influencing compliance behaviour. METHODS: Randomised double-blind intervention study on the effect of infant formulas with reduced allergenicity in healthy, term newborns at risk of atopy. Maternal compliance with dietary recommendations concerning milk and solid food feeding was categorised. RESULTS: A total of 2252 newborns were randomised to one of four study formulas. The drop out rate during the first year of life was 13.5% (n = 304). The rates of high, medium, and low compliance to milk feeding during weeks 1-16 were 83.4%, 4.0%, and 7.5%; the corresponding rates to solid food feeding during weeks 1-24 were 60.0%, 12.1%, and 22.9%. In 5.1% of subjects no nutritional information was available. Low compliance was more frequent among non-German parents, parents with a low level of education, young mothers, smoking mothers, and those who weaned their infant before the age of 2 months. CONCLUSIONS: Evaluation of allergy preventive programmes should take into account non-compliance for assessing the preventive effectiveness on study outcome.

Pets and vermin are associated with high endotoxin levels in house dust.

BACKGROUND: Previous studies have shown that the risk for allergic sensitization is lower in children who grew up on farms and in young adults who were exposed to dogs in early childhood. A higher microbial exposure in general and in particular to endotoxin in early childhood might contribute to this lower risk of atopy. OBJECTIVE: We examined whether the presence of pets or vermin in the home is associated with higher endotoxin concentrations in settled house dust. METHODS: House dust was sampled in a standardized manner on the living room floors of 454 homes of German children aged 5-10 years (participation rate 61%). Endotoxin was assessed with a quantitative kinetic chromogenic Limulus Amebocyte Lysate (LAL) method. Associations between endotoxin levels, pets and vermin are presented as ratios of the crude and confounder adjusted geometric means (means ratios) in the category of study vs. a reference category using multiple linear regression models. RESULTS: Endotoxin concentrations in living room floor dust sampled in homes without pets and vermin were lower (1246 ng per square meter, 1519 ng endotoxin/g dust, n = 157) than those sampled in homes with pets or vermin (2267 ng per square meter, 2200 ng endotoxin/g dust, n = 296). After adjustment for city of residence, season of dust sampling, age of the building and story of the dwelling, means ratios for endotoxin expressed per gram of dust were statistically significantly increased for dog (1.64, 95% CI 1.09-2.46), for cat (1.50, 95% CI 1.03-2.18) and for cockroach (3.01, 95% CI 1.37-6.60), whereas no major statistically significant associations were found for other pets, ants and mice. CONCLUSION: Keeping a dog or a cat in the home is consistent with higher exposure to endotoxin and might therefore contribute to the lower risk of atopy in later life.

Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.

While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.

Transcriptional activation by the human Hsp70-associating protein Hap50.

We investigated human Hap50, the large isoform of the previously characterized Hsp70/Hsc70-associating protein Hap46, also called BAG-1, for effects on transcriptional activities. Overproduction by transient transfection led to enhanced expression of reporter gene constructs in various cell types using different promoters, suggesting independence of promoter type. Similarly, overexpression of Hap50 resulted in increased levels of poly(A)(+ )mRNAs in HeLa, COS-7, 3T3 and HTC cells. Concomitantly, the expression of some selected endogenous genes, such as those coding for c-Jun and the glucocorticoid receptor, was enhanced significantly relative to actin. Nuclear runoff transcription assays using HeLa cells showed that the effect is caused by increased transcription rates rather than mRNA stabilization. Activation of transcription by Hap50 occurred at 37 degrees C and did not require prior thermal stress, as is the case for Hap46. In accordance with these biological effects, Hap50 is localized exclusively in the nuclear compartment of different cell types, whereas Hap46 is mostly cytoplasmic in unstressed cells, as revealed by use of fusion constructs with green fluorescent protein. High cellular levels of Hap50 were found to make cells less susceptible to adverse environmental effects such as heat stress. Our data suggest that Hap50 is a nuclear protein that acts in cells to increase the transcription of various genes.

Dissection of the ATP-binding domain of the chaperone hsc70 for interaction with the cofactor Hap46.

Several unrelated proteins are known that specifically interact with members of the mammalian hsp70 chaperone protein family independent of the hsp70 substrate-binding site. One of these is Hap46, also called BAG-1, which binds to the ATP-binding domain of hsp70 and its constitutively expressed, highly homologous counterpart hsc70, thereby affecting nucleotide binding, as well as protein folding properties, of these molecular chaperones. In an attempt to delineate the potential contact sites on hsp70/hsc70 involved in this interaction we made use of the following two independent approaches: (i) screening of membrane-bound peptide libraries based on the sequence of the ATP-binding domain and (ii) the phage-display technique with random dodecapeptides. These approaches yielded partially overlapping results and identified several possible contact regions. On the space-filling model of hsc70, the two major contact areas for Hap46 delineated in the present study are located on the same side of the molecule on either subdomain that border the central cleft harboring the nucleotide-binding site. We suggest that this bridging affects the conformation of the ATP-binding domain in a way similar to the opening of the nucleotide-binding cleft produced in the bacterial hsp70 homologue DnaK upon binding its regulatory protein GrpE.

The hsp70-associating protein Hap46 binds to DNA and stimulates transcription.

We investigated the ubiquitously expressed hsp70-associating protein Hap46, which is also called RAP46 and is homologous to BAG-1, for activities independent of hsp70 interactions. We observed in vitro binding to various DNA fragments but detected no apparent sequence specificity. Deletion of the amino-terminal decapeptide, which contains two clusters of three basic amino acids each, abolished the DNA-binding ability of Hap46. Similarly, exchange of either of these positively charged clusters for three alanines resulted in loss of DNA binding. Using a fusion of Hap46 and green fluorescent protein, we found preferential accumulation in cell nuclei on heat stress as compared with unstressed cells. The repressive effect of heat shock on overall transcriptional activity in human DU145 carcinoma cells was largely prevented when Hap46 was overexpressed by transfection. Such overproduction of Hap46 also resulted in enhanced expression of specific reporter gene constructs and in increased levels of mRNAs specific for hsp70 and hsp40 after temperature stress. In vitro transcription with nuclear extracts was stimulated greatly by Hap46. Like DNA binding, transcriptional enhancement required amino-terminally located basic amino acid residues but not the carboxyl-terminal portion of Hap46 known to participate in hsp70 interaction. Our results show that Hap46 is a bifunctional protein that can interact with both hsp70s and DNA, employing different portions of the molecule. They also suggest that Hap46 is involved in temperature-sensitive regulation of transcription, acting as a general transcriptional activator.

Interference between proteins Hap46 and Hop/p60, which bind to different domains of the molecular chaperone hsp70/hsc70.

Several structurally divergent proteins associate with molecular chaperones of the 70-kDa heat shock protein (hsp70) family and modulate their activities. We investigated the cofactors Hap46 and Hop/p60 and the effects of their binding to mammalian hsp70 and the cognate form hsc70. Hap46 associates with the amino-terminal ATP binding domain and stimulates ATP binding two- to threefold but inhibits binding of misfolded protein substrate to hsc70 and reactivation of thermally denatured luciferase in an hsc70-dependent refolding system. By contrast, Hop/p60 interacts with a portion of the carboxy-terminal domain of hsp70s, which is distinct from that involved in the binding of misfolded proteins. Thus, Hop/p60 and substrate proteins can form ternary complexes with hsc70. Hop/p60 exerts no effect on ATP and substrate binding but nevertheless interferes with protein refolding. Even though there is no direct interaction between these accessory proteins, Hap46 inhibits the binding of Hop/p60 to hsc70 but Hop/p60 does not inhibit the binding of Hap46 to hsc70. As judged from respective deletions, the amino-terminal portions of Hap46 and Hop/p60 are involved in this interference. These data suggest steric hindrance between Hap46 and Hop/p60 during interaction with distantly located binding sites on hsp70s. Thus, not only do the major domains of hsp70 chaperones communicate with each other, but cofactors interacting with these domains affect each other as well.

The chaperone cofactor Hop/p60 interacts with the cytosolic chaperonin-containing TCP-1 and affects its nucleotide exchange and protein folding activities.

The folding of protein structures often requires the presence of molecular chaperones and/or chaperonin complexes. We here investigated the inhibitory effects of the chaperone cofactors Hop/p60 and Hap46. By coimmunoprecipitation, we observed a direct interaction of the eukaryotic chaperonin-containing TCP-1 (CCT) purified from rabbit reticulocyte lysate with Hop/p60. By contrast, Hap46 was not coprecipitated. Binding of Hop/p60 to CCT is dependent on the presence of ATP or ADP and occurs through carboxyl-terminal sequences of Hop/p60. Hop/p60 significantly stimulates nucleotide exchange on CCT but not its ATPase activity, while Hap46 has no effects. We used denatured firefly luciferase as a model protein and found decreased binding to CCT in the presence of Hop/p60 and ATP. This coincides with the inhibitory effect of Hop/p60 on luciferase reactivation in an assay using purified CCT in combination with hsc70 and hsp40. We also observed that an antibody directed against one of the subunits of CCT efficiently inhibits refolding in a system which depends on crude reticulocyte lysate.

RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis.

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.

Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity.

We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy-terminal domain of hsp70 while Hap-46 and Hip/p48 bind the 383 residue amino-terminal ATP binding domain. Hap-46 and Hip/p48 competed for binding to hsc70, while Hap-46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70. Hap-46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60. These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins.

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.

The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin.

The ansamycin antibiotic geldanamycin, which specifically interacts with the heat shock protein hsp90, was used to study the function of hsp90 in steroid hormone receptors. We observed inhibition of glucocorticoid-specific gene induction in several responsive cell systems. Hormone binding abilities of receptors for glucocorticoid, progestin, androgen, and estrogen were inhibited upon exposing intact cells to geldanamycin. Inhibition was only seen when geldanamycin was applied to cell cultures under growth conditions or was present during in vitro synthesis; presynthesized receptors in cell extracts were not affected. Upon withdrawal of geldanamycin, glucocorticoid binding ability was regained; this was partially independent of de novo protein synthesis. Geldanamycin caused decreased levels of immunoreactive glucocorticoid receptors in wild-type cells with enhanced degradation occurring through the ubiquitin-proteasome pathway. Analysis of receptors from treated cells revealed a heteromeric structure of normal size in which the receptor polypeptide is complexed with normal amounts of hsp90 and the immunophilin p59. These data support the view that hsp90 actively participates in steroid-induced signal transduction, and they suggest that geldanamycin affects receptor action without disrupting hsp90-containing heterocomplexes per se. Nevertheless, complexes synthesized and assembled in vitro in the presence of geldanamycin differ from receptors of cellular origin.