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Childhood leukemia is the most common cancer diagnosed in children worldwide. However, only a few causes have been established so far, mainly some genetic syndromes and high doses of ionizing radiation. Major efforts have been undertaken to study the relationship between environmental factors and the risk of childhood leukemia, inspired by geographical variation in incidence rates. Some evidence has emerged for parental occupational exposures to pesticides, whereas there is less evidence for an association with postnatal pesticide exposure. Diagnostic radiation and radon exposure have been suggested but there remains a lack of convincing studies. Extremely low-frequency magnetic fields consistently showed a small increase in risk in numerous studies, but bias and confounding cannot be ruled out as possible explanations. From among factors other than environmental and radiation-related, the most promising candidate is abnormal patterns to common infections, but which children are most at risk and the pathways are not fully understood. In conclusion, although childhood leukemia shows some distinct incidence patterns by sex, age, and geography suggesting a role of the environment in its etiology, no major environmental risk factors including radiation have been established as major contributors to the global childhood leukemia burden. Due to the young age at diagnosis and evidence of chromosomal damage before birth in many of the affected children, parental exposures remain of high interest. Although cure rates of childhood leukemia are high in economically developed countries, because of the adverse late effects of the disease and its treatment, identification of modifiable risk factors for implementing primary prevention remains the ultimate goal., (Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.)

The determination of Δ(9)-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in hair is a major routine task in forensic laboratories worldwide. A comprehensively automated liquid-liquid extraction (LLE) method has been developed. The automation was carried out by an x-y-z sample robot equipped with modules capable of shaking, centrifugation and solvent evaporation. It comprises digestion of hair in sodium hydroxide solution, LLE, extract evaporation, reconstitution in silylation reagent, inlet derivatization and GC-MS analysis. Method validation guidelines of the Society for Toxicological and Forensic Chemistry were fulfilled. The limit of quantification (LOQ) was 0.01 ng/mg for THC, 0.06 ng/mg for CBN and 0.03 ng/mg for CBD. This is below the required LOQ for THC (0.02 ng/mg) in medical psychological assessments in Germany. Also it is far below the required LOQ of the Society of Hair Testing of 0.1 ng/mg for THC. Four-round robin tests were passed successfully and several post- and ante-mortem samples were analyzed. To date the method is routinely employed at the Institute of Legal Medicine in Giessen, Germany. To the best of our knowledge, this is the first publication on a comprehensively automated classical LLE workflow in the field of hair analysis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Due to diverse findings as to the role of family factors for childhood cancer survival even within Europe, we explored a nationwide, register-based cohort of Danish children with hematological malignancies., Methods: All children born between 1973 and 2006 and diagnosed with a hematological malignancy before the age of 20 years (N = 1,819) were followed until 10 years from diagnosis. Kaplan-Meier curves and Cox proportional hazards models estimating hazard ratios (HR) and 95% confidence intervals (CI) were used to assess the impact of family characteristics on overall survival in children with hematological malignancies., Results: Having siblings and increasing birth order were associated with reduced survival from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Associations with AML were strongest and statistically significant. HRs of 1.62 (CI 0.85; 3.09) and 5.76 (CI 2.01; 16.51) were observed for the fourth or later born children with ALL (N = 41) and AML (N = 9), respectively. Children with older parents showed a tendency toward inferior ALL survival, while for AML young maternal age was related to poorer survival. Based on small numbers, a trend toward poorer survival from non-Hodgkin lymphoma was observed for children having siblings and for children of younger parents., Conclusions: Further research is warranted to gain further knowledge on the impact of family factors on childhood cancer survival in other populations and to elaborate potential underlying mechanisms and pathways of those survival inequalities., (© 2016 Wiley Periodicals, Inc.)

Background One fifth of all deaths among children in Europe are accounted for by cancer. If this is to be reduced there is a need for studies on not only biology and treatment approaches but also on how social factors influence cure rates. We investigated how various socioeconomic characteristics were associated with survival after childhood cancer. Material and methods In a nationwide cohort of 3797 children diagnosed with cancer [hematological cancer, central nervous system (CNS) tumors, non-CNS solid tumors] before age 20 between 1990 and 2009 we identified parents and siblings and obtained individual level parental socioeconomic variables and vital status through 2012 by linkage to population-based registries. Hazard ratios (HR) and 95% confidence intervals (CI) for dying were estimated using multivariate Cox proportional hazard models. Results For all children with cancer combined, survival was slightly but not statistically significantly better the higher the education of the mother or the father, and with maternal income. Significantly better survival was observed when parents were living together compared to living alone and worse survival when the child had siblings compared to none. Young (<20) or older (≥40) maternal age showed non-significant associations, but based on small numbers. For hematological cancers, no significant associations were observed. For CNS tumors, better survival was seen with parents living together (HR 0.70, CI 0.51-0.97). For non-CNS solid tumors, survival was better with high education of the mother (HR 0.66, CI 0.44-0.99) compared to basic and worse for children with one (HR 1.45, CI 1.11-1.89) or two or more siblings (HR 1.29, CI 0.93-1.79) (p for trend 0.02) compared to none. Conclusion The impact of socioeconomic characteristics on childhood cancer survival, despite equal access to protocolled and free-of-charge treatment, warrants further and more direct studies of underlying mechanisms in order to target these as a means to improve survival rates.

The analysis of Δ(9)-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver's licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 μg/L, for 11-OH-THC were 0.1 and 0.8 μg/L, and for THC-COOH were 0.3 and 1.1 μg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 μg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis. Graphical abstract GC/MS with MPS Dual Head at the Institute of Legal Medicine, Giessen, Germany. Modules from left to right: (quick) Mix (for LLE), wash station, tray 1 (vials for extracts), solvent reservoir, (m) VAP (for extract evaporation), Solvent Filling Station (solvent supply), cooled tray 2 (vials for serum samples), and centrifuge (for phase separation).

Little is known about the incidence patterns of hematologic malignancies in Sub-Saharan Africa, including South Africa. We estimated incidence rates of pathology-confirmed adult cases of leukemia, myeloma and related diseases (myeloma), Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL) reported to the National Cancer Registry of South Africa (NCR) between 2000 and 2006, by age, gender, and population group (Black, White, Coloured, Asian/Indian). Gender-specific age-standardized rates were calculated overall and by population group and incidence rate ratios (IRRs) were estimated using Poisson regression models. Between 2000 and 2006, there were 14662 cases of leukemia, myeloma, HL, and NHL reported to the registry. Incidence rates of reported hematologic malignancies were generally 20-50% higher among males than females. Our analyses suggested marked differences in the rates of reported hematologic malignancies by population group which were most pronounced when comparing the White versus Black population groups (IRRs ranging from 1.6 for myeloma to 3.8 for HL for males and females combined). Challenges related to diagnosis and reporting of cancers may play a role in the patterns observed by population group while the set-up of the NCR (pathology-based) could lead to some degree of under-ascertainment in all groups. This is the first country-wide report of the incidence of hematologic malignancies in South Africa. Despite challenges, it is important to analyze and report available national cancer incidence data to raise awareness of the cancer burden and to characterize patterns by demographic characteristics so as ultimately to improve the provision of cancer-related health care., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification. In ARIMMORA for the first time, a transgenic mouse model was used to mimic the most common childhood leukemia: new pathogenic mechanisms were indicated, but more data are needed to draw definitive conclusions. Although experiments in different animal strains showed exposure-related decreases of CD8+ T-cells, a role in carcinogenesis must be further established. No direct damage of DNA by exposure was observed. Overall in the literature, there is limited evidence of carcinogenicity in humans and inadequate evidence of carcinogenicity in experimental animals, with only weak supporting evidence from mechanistic studies. New exposure data from ARIMMORA confirmed that if the association is nevertheless causal, up to 2% of childhood leukemias in Europe, as previously estimated, may be attributable to ELF-MF. In summary, ARIMMORA concludes that the relationship between ELF-MF and childhood leukemia remains consistent with possible carcinogenicity in humans. While this scientific uncertainty is dissatisfactory for science and public health, new mechanistic insight from ARIMMORA experiments points to future research that could provide a step-change in future assessments. Bioelectromagnetics. 37:183-189, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Results from case-control studies have shown an increased risk of acute lymphoblastic leukaemia (ALL) in young children born by caesarean delivery, and prelabour caesarean delivery in particular; however, an association of method of delivery with childhood leukaemia subtypes has yet to be established. We therefore did a pooled analysis of data to investigate the association between childhood leukaemia and caesarean delivery., Methods: We pooled data from 13 case-control studies from the Childhood Leukemia International Consortium done in nine countries (Canada, Costa Rica, Egypt, France, Germany, Greece, Italy, New Zealand, and the USA) for births from 1970-2013. We analysed caesarean delivery overall and by indications that probably resulted in prelabour caesarean delivery or emergency caesarean delivery. We used multivariable logistic regression models, adjusted for child's birthweight, sex, age, ethnic origin, parental education, maternal age, and study, to estimate odds ratios (ORs) and 95% CIs for the risk of ALL and acute myeloid leukaemia (AML) in children aged 0-14 years at diagnosis., Findings: The studies provided data for 8780 ALL cases, 1332 AML cases, and 23 459 controls, of which the birth delivery method was known for 8655 (99%) ALL cases, 1292 (97%) AML cases, and 23 351 (>99%) controls. Indications for caesarean delivery were available in four studies (there were caesarean deliveries for 1061 of 4313 ALL cases, 138 of 664 AML cases, and 1401 of 5884 controls). The OR for all indications of caesarean delivery and ALL was 1·06 (95% CI 0·99-1·13), and was significant for prelabour caesarean delivery and ALL (1·23 [1·04-1·47]; p=0·018). Emergency caesarean delivery was not associated with ALL (OR 1·02 [95% CI 0·81-1·30]). AML was not associated with caesarean delivery (all indications OR 0·99 [95% CI 0·84-1·17]; prelabour caesarean delivery 0·83 [0·54-1·26]; and emergency caesarean delivery 1·05 [0·63-1·77])., Interpretation: Our results suggest an increased risk of childhood ALL after prelabour caesarean delivery. If this association is causal, maladaptive immune activation due to an absence of stress response before birth in children born by prelabour caesarean delivery could be considered as a potential mechanism., Funding: National Cancer Institute., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, a series of new benzohydroxamates were prepared as potent inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by these 3-amidobenzohydroxamates. The newly designed inhibitors were evaluated in screens for enzyme inhibitory activity against schistosome and human HDACs. Twenty-seven compounds were found to be active in the nanomolar range, and some of them showed selectivity toward smHDAC8 over the major human HDACs (1 and 6). The active benzohydroxamates were additionally screened for lethality against the schistosome larval stage using a fluorescence-based assay. Four of these showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.

The use of cationic lipids as gene delivery systems is a basic method in gene therapy. Through ongoing research, lipofection is currently the leader of non-viral vectors in clinical trials. However, in order to unleash the full potential of lipofection further intensive investigations are indispensable. In this study, various lipoplex formulations were compared regarding their ability to bind DNA. To obtain information about a possible premature release of DNA at the cell surface, heparin and chondroitin dependent lipoplex destabilization experiments were carried out. Complementary investigations in cell culture were performed to quantify DNA outside the cell. Additionally, DNase I stability was investigated. In this regard a multitude of methods, namely confocal laser scanning microscopy (CLSM), polymerase chain reaction (PCR), cell culture experiments, ethidium bromide assay, gel electrophoresis, Langmuir-isotherm experiments, infrared reflection absorption spectroscopy (IRRAS), Brewster angle microscopy (BAM), zeta-(ζ)-potential measurements, and dynamic light scattering (DLS), were applied. Although the complexation of DNA is a fundamental step, we show that the DNA release by biological agents (proteoglycans) and an unsuccessful cell attachment are major transfection limiting parameters., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ultraviolet radiation (UVR) is part of the electromagnetic spectrum emitted naturally from the sun or from artificial sources such as tanning devices. Acute skin reactions induced by UVR exposure are erythema (skin reddening), or sunburn, and the acquisition of a suntan triggered by UVR-induced DNA damage. UVR exposure is the main cause of skin cancer, including cutaneous malignant melanoma, basal-cell carcinoma, and squamous-cell carcinoma. Skin cancer is the most common cancer in fair-skinned populations, and its incidence has increased steeply over recent decades. According to estimates for 2012, about 100,000 new cases of cutaneous melanoma and about 22,000 deaths from it occurred in Europe. The main mechanisms by which UVR causes cancer are well understood. Exposure during childhood appears to be particularly harmful. Exposure to UVR is a risk factor modifiable by individuals' behaviour. Excessive exposure from natural sources can be avoided by seeking shade when the sun is strongest, by wearing appropriate clothing, and by appropriately applying sunscreens if direct sunlight is unavoidable. Exposure from artificial sources can be completely avoided by not using sunbeds. Beneficial effects of sun or UVR exposure, such as for vitamin D production, can be fully achieved while still avoiding too much sun exposure and the use of sunbeds. Taking all the scientific evidence together, the recommendation of the 4th edition of the European Code Against Cancer for ultraviolet radiation is: "Avoid too much sun, especially for children. Use sun protection. Do not use sunbeds.", (Copyright © 2015 International Agency for Research on Cancer. Published by Elsevier Ltd. All rights reserved.)

Ionising radiation can transfer sufficient energy to ionise molecules, and this can lead to chemical changes, including DNA damage in cells. Key evidence for the carcinogenicity of ionising radiation comes from: follow-up studies of the survivors of the atomic bombings in Japan; other epidemiological studies of groups that have been exposed to radiation from medical, occupational or environmental sources; experimental animal studies; and studies of cellular responses to radiation. Considering exposure to environmental ionising radiation, inhalation of naturally occurring radon is the major source of radiation in the population - in doses orders of magnitude higher than those from nuclear power production or nuclear fallout. Indoor exposure to radon and its decay products is an important cause of lung cancer; radon may cause approximately one in ten lung cancers in Europe. Exposures to radon in buildings can be reduced via a three-step process of identifying those with potentially elevated radon levels, measuring radon levels, and reducing exposure by installation of remediation systems. In the 4th Edition of the European Code against Cancer it is therefore recommended to: "Find out if you are exposed to radiation from naturally high radon levels in your home. Take action to reduce high radon levels". Non-ionising types of radiation (those with insufficient energy to ionise molecules) - including extremely low-frequency electric and magnetic fields as well as radiofrequency electromagnetic fields - are not an established cause of cancer and are therefore not addressed in the recommendations to reduce cancer risk., (Copyright © 2015 International Agency for Research on Cancer. Published by Elsevier Ltd. All rights reserved.)

Purpose: Many studies have investigated the possible association between birth order and risk of childhood cancer, although the evidence to date has been inconsistent. Birth order has been used as a marker for various in utero or childhood exposures and is relatively straightforward to assess., Methods: Data were obtained on all children born in Denmark between 1973 and 2010, involving almost 2.5 million births and about 5,700 newly diagnosed childhood cancers before the age of 20 years. Data were analyzed using Poisson regression models., Results: We failed to observe associations between birth order and risk of any childhood cancer subtype, including acute lymphoblastic leukemia; all rate ratios were close to one. Further analyses stratified by birth cohort (those born between 1973 and 1990, and those born between 1991 and 2010) also failed to show any associations. Considering stillbirths and/or controlling for birth weight and parental age in the analyses had no effect on the results. Analyses by years of birth (those born between 1973 and 1990, and those born between 1991 and 2010) did not show any changes in the overall pattern of no association., Conclusions: In this large cohort of all children born in Denmark over an almost 40-year period, we did not observe an association between birth order and the risk of childhood cancer.

Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Higher childhood cancer incidence rates are generally reported for high income countries although high quality information on descriptive patterns of childhood cancer incidence for low or middle income countries is limited, particularly in Sub-Saharan Africa. There is a need to quantify global differences by cancer types, and to investigate whether they reflect true incidence differences or can be attributed to under-diagnosis or under-reporting. For the first time, we describe childhood cancer data reported to the pathology report-based National Cancer Registry of South Africa in 2000-2006 and compare our results to incidence data from Germany, a high income country. The overall age-standardized incidence rate (ASR) for South Africa in 2000-2006 was 45.7 per million children. We observed substantial differences by cancer types within South Africa by racial group; ASRs tended to be 3-4-fold higher in South African Whites compared to Blacks. ASRs among both Black and White South Africans were generally lower than those from Germany with the greatest differences observed between the Black population in South Africa and Germany, although there was marked variation between cancer types. Age-specific rates were particularly low comparing South African Whites and Blacks with German infants. Overall, patterns across South African population groups and in comparison to Germans were similar for boys and girls. Genetic and environmental reasons may probably explain rather a small proportion of the observed differences. More research is needed to understand the extent to which under-ascertainment and under-diagnosis of childhood cancers drives differences in observed rates., (© 2014 UICC.)

Previously, a glycoglycerolipid isolated from marine algae was reported to be a potent and selective inhibitor of the human Myt1 kinase, an enzyme involved in cell cycle regulation with great potential as an anti-cancer target. Based on that report, a lot of research effort has been invested by several working groups to synthesize and derivatize this compound. However, reliable assay data confirming the inhibitory potential and the mechanism of action of these glycoglycerolipids are missing so far. Here, based on experimental data and theoretical considerations, we show that the aforesaid glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6'-amino-6'-deoxy-α-d-glucosyl)-sn-glycerol is not an inhibitor of the human Myt1 kinase.

Background: Little is known about the relationship between family characteristics and survival from childhood acute lymphoblastic leukaemia (ALL), which we studied for the first time in German children., Methods: ALL cases were diagnosed between 1992 and 1994 and information on family characteristics was collected during a previously conducted nationwide case-control study. Children were followed for 10 years after diagnosis, as few disease-related events occur afterwards. Cox proportional hazards models estimating hazard ratios (HR) were calculated using overall as well as event-free survival methods., Results: Second born children showed statistically significant better survival compared to first or later born children, with HRs ranging between 0.54 and 0.64 compared to firstborns. Somewhat poorer survival was observed for children having 3 or more siblings. A relationship was found for parental age at child's diagnosis, with poorer survival for children with younger parents (≤25 years of age at child's diagnosis), or with older fathers. The HR was statistically significant for fathers being ≥41years of age (HR of 2.1). No relationship between degree of urbanization of the place of residence at diagnosis and ALL survival was observed., Conclusion: Family circumstances may have an impact on survival from childhood ALL in Germany. Further research is warranted to elaborate the relationship of specific family characteristics and ALL survival and to investigate possible differential adherence to therapy and interactions with physicians., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The neuropeptide S (NPS) system was discovered as a novel neurotransmitter system a decade ago and has since been identified as a key player in the modulation of fear and anxiety. Genetic variations of the human NPS receptor (NPSR1) have been associated with pathologies like panic disorders. However, details on the molecular fundamentals of NPSR1 activity in neurons remained elusive. We expressed NPSR1 in primary hippocampal cultures. Using single-cell calcium imaging we found that NPSR1 stimulation induced calcium mobilization from the endoplasmic reticulum via activation of IP3 and ryanodine receptors. Store-operated calcium channels were activated in a downstream process mediating entry of extracellular calcium. We provide the first detailed analysis of NPSR1 activity and the underlying intracellular pathways with respect to calcium mobilization in neurons.

The authors report on the death of two women who were married to the same man one after the other. Exhumation and toxicological investigation of the first wife, who had died 7 years before, did not produce any conclusive evidence of homicide. With regard to the circumstances of death of the second wife the husband made different statements. According to the result of the chemical and toxicological investigations death was caused by acute intoxication with the beta-blocker metoprolol. The man was found guilty of killing on request (which is a criminal offence in Germany) by administering the beta-blocker metoprolol through a transnasal gastric tube.

Background: Sex, age, immunophenotype and white blood cell count at diagnosis are well accepted predictors of survival from acute lymphoblastic leukaemia (ALL) in children. Less is known about the relationship between socio-economic determinants and survival from paediatric ALL, studied here for the first time in German children., Methods: ALL cases were diagnosed between 1992 and 1994 and their parents interviewed during a previous nationwide case-control study. Children were followed-up for 10 years after diagnosis by the German Childhood Cancer Registry. Cox proportional hazards models estimating hazard ratios (HRs) were calculated to assess the impact of selected socio-demographic characteristics on overall and event-free survival., Results: Overall survival was 82.5%, with a higher proportion of girls than boys surviving (85% versus 81%). We found a non-linear relationship between age at diagnosis and survival, with poorer survival in infants and children aged >5 years. There was no association between socio-economic factors and survival or risk of relapse. For five levels of increasing family income, all HRs were close to one. No relationship was seen with parental educational level., Conclusion: Socio-economic determinants did not affect ALL survival in West German children, in contrast to studies from some other countries. Dissimilarities in social welfare systems, including access to health care, lifestyle and differences in treatment may contribute to these differences in findings. Our observation of no social inequalities in paediatric ALL survival is reassuring, but needs continued monitoring to assess the potential impact of evolvement of treatment options and changes in paediatric health service., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Identification of compounds that can bind to a target protein with high affinity is a nontrivial task in structure-based drug design. Several approaches ranging from simple scoring methods to more computationally demanding methods are usually applied for this purpose. In the current work, we used ligand docking in combination with QM/MM-GBSA, MM-GBSA, and MM-PBSA rescoring to discriminate between active and inactive Myt1 kinase inhibitors. Results show that QM/MM-GBSA rescoring performs better than normal docking scores or MM-GBSA rescoring in classifying active and inactive inhibitors. We also applied QM/MM-GBSA rescoring to estimate the binding affinities of compounds from different virtual screening runs. To prove our approach and to confirm its predictive power, a few compounds which were predicted to be active were purchased and experimentally tested. Among the five selected compounds, three showed significant inhibition of recombinant Myt1. PD-173952, which yielded a favorable QM/MM-GBSA binding free energy, showed a K(i) value of 8.1 nM. In addition, two compounds, PD-180970 and saracatinib, showed inhibition at the low micromolar level. Thus, the developed protocol might be useful for further virtual screening experiments to better discriminate between active and inactive compounds and to further optimize the identified hits.

Abstract The human Myt1 kinase is a regulator of Cdk1/CycB and, hence, important for the G2/M transition in the cell cycle. It may act as a target for drug development, but suitable assay systems for assessing potential inhibitors are lacking so far. Herein, we describe the rational development of a fluorescence anisotropy-based kinase binding assay. A suitable fluoroprobe based on the tyrosine kinase inhibitor dasatinib was synthesized and tested with Myt1 and several other kinases for control purposes. The probe acted as expected in terms of specificity and reversibility, and a Myt1 assay was set up. Notwithstanding the moderate Kd of the starting compound dasatinib before chemical modification, satisfying Z' factors >0.5 were achieved. A validation with known kinase inhibitors demonstrated the applicability of the assay and led to a reliable ranking of the tested active compounds.

In the human cell cycle, the Myt1 kinase is a crucial regulator of the G2/M transition. Because this membrane-associated kinase is hard to obtain and assay, there is a distinct lack of data so far. Here we report the derivatization of a glycoglycerolipid which was shown previously to be active in a Myt1 activity assay. These compounds were tested in a binding assay together with a set of common kinase inhibitors against a full-length Myt1 expressed in a human cell line. Dasatinib exhibited nanomolar affinity whereas broad coverage inhibitors such as sunitinib and staurosporine derivatives did not show any effect. We also carried out docking studies for the most potent compounds allowing further insights into the inhibitor interaction of this kinase. The glycoglycerolipids showed no significant effects in the binding assay, endorsing the idea of a mechanism of action distant from the active site., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The incidence of cutaneous malignant melanoma has steadily increased over the past 50 years in predominately fair-skinned populations. This increase is reported to have leveled off recently in several Northern and Western European countries, Australia, New Zealand and in North America. We studied the global patterns and time trends in incidence of melanoma by country and sex, with a focus on and age- and cohort-specific variations. We analyzed the incidence data from 39 population-based cancer registries, examining all-ages and age-truncated standardized incidence rates of melanoma, estimating the annual percentage change and incidence rate ratios from age-period-cohort models. Incidence rates of melanoma continue to rise in most European countries (primarily Southern and Eastern Europe), whereas in Australia, New Zealand, the U.S., Canada, Israel and Norway, rates have become rather stable in recent years. Indications of a stabilization or decreasing trend were observed mainly in the youngest age group (25-44 years). Rates have been rising steadily in generations born up to the end of the 1940s, followed by a stabilization or decline in rates for more recently born cohorts in Australia, New Zealand, the U.S., Canada and Norway. In addition to the birth cohort effect, there was a suggestion of a period-related influence on melanoma trends in certain populations. Although our findings provide support that primary and secondary prevention can halt and reverse the observed increasing burden of melanoma, they also indicate that those prevention measures require further endorsement in many countries., (Copyright © 2012 UICC.)

The human Myt1 kinase (PKMYT1) is an important regulator of the G2/M transition in the cell cycle. Presently, limited knowledge about its substrate recognition is available. Here, various potential substrates were investigated by different antibody based techniques including fluorescence polarization immunoassays and immunoblotting. Regarding both Thr and Tyr kinase activity, only protein substrates were found to be phosphorylated by Myt1, whereas any tested peptide was not recognized. In silico molecular dynamics studies were used to compare the stability of the Myt1 peptide complex with Wee1 peptide complex and support the biochemical findings. Furthermore, a Myt1 kinase binding assay suggests Myt1 being insensitive to staurosporine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis and in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous pathway for transporting newly synthesized polypeptides into the ER lumen and may also allow calcium leakage from the ER into the cytosol. In this study, planar lipid bilayer experiments demonstrated that the Sec61 complex is permeable to calcium ions. We also investigated whether silencing the SEC61A1 gene affected calcium leakage from the ER. Silencing the SEC61A1 gene using two different siRNAs in HeLa cells for 96 hours had little effect on cell growth and viability. However, calcium leakage from the ER was greatly decreased in the SEC61A1-silenced cells. Thus, the Sec61 complexes that are present in the ER membrane of nucleated cells form calcium leak channels that may play a crucial role in calcium homeostasis.

An analytical procedure for the simultaneous determination in human serum of 43 common drugs of abuse and their metabolites belonging to the different chemical and toxicological classes of amphetamines, benzodiazepines, dibenzazepines, cocaine, lysergic acid diethylamide, opioids, phencyclidine, tricyclic antidepressants, and zolpidem, using 33 deuterated standards, is presented. The sample treatment was developed to be a very simple protein precipitation and filtration. All analyses were performed with a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in positive ionization mode. All analytes were calibrated up to 550 μg/L. The limit of detection ranged from 0.6 ng/mL (EDDP) to 13.7 ng/mL (flunitrazepam). The method has been validated according to the guidelines of the Gesellschaft für Toxikologische und Forensische Chemie, using three multiple reaction mode (MRM) transitions and retention time for positive compound identification, instead of two MRMs, in anticipation of the new guidelines for January 2011.

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

Using light to tune the activity of proteins represents a very attractive avenue for creating various temporal interferences in living systems. In this mini-review, we highlight a few recent developments in this broad and exciting field. Among the various methods, we have discussed in more detail the advantages and future challenges in using light switchable drugs to regulate the signaling proteins in the immune system.

The human FK506-binding protein 38 (FKBP38) regulates Bcl-2 in neuronal apoptosis. To control Bcl-2 activity, FKBP38 requires a prior interaction with the Ca(2+)-sensor calmodulin (CaM). The resulting FKBP38/CaM complex is unique within the FKBP family. Here, we present novel insights into the structural arrangement of this complex. Chemical shift perturbation analyses of the individual protein domains revealed two separate interaction sites between FKBP38 and CaM. On the one hand, residues Glu303, Tyr307 and Leu311, belonging to the predicted CaM-binding site at the C-terminal end of FKBP38, become embedded in the hydrophobic target protein-binding cleft of the C-terminal CaM lobe. On the other hand, in a second binding interaction, the N-terminal end of the catalytic FKBP38 domain shows surface contacts to the AB and CD loops of CaM as well as the adjacent helices. Furthermore, a Glu-rich region at the non-structured FKBP38 N-terminus features additional contacts to CaM helix A. In combination with previous results, we thus conclude that the FKBP38/CaM complex is constituted by (i) a Ca(2+)-dependent interaction of the CaM-binding motif at the C-terminal end of FKBP38 with the C-terminal CaM lobe and (ii) a Ca(2+)-independent interaction between the N-terminal CaM lobe and the N-terminal region of the catalytic FKBP38 domain.

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D(lat) = 10-25 mum(2)/s and rotational diffusion coefficients ranging from D(rot) = 2.8 - 1.4 x 10(7) s(-1) were measured in pure liquid-disordered (L(d)) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L(o)) domains segregated from the L(d) phase at 23 degrees C. The lateral mobility of lipids in L(o) domains was around eightfold lower compared to those in the L(d) phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L(o) phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L(o) domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L(d) phase., ((c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, protein translocation across and insertion into the membrane of the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex or translocon. In our previous electrophysiological studies, we characterized the mammalian Sec61 channel from Canis familiaris. Here we extended these initial results to the Sec61 channel from the yeast Saccharomyces cerevisiae and compared the basic electrophysiological properties of both channel preparations with respect to the gating behaviour, distribution of channel open states, ionic conductance, approximated pore dimensions, reversal potential and selectivity as well as voltage-dependent open probability. We found that the Sec61 complexes from both species displayed conformable characteristics of the highly dynamic channel in an intrinsically open state. In contrast, the bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, displayed under the same experimental conditions significantly different properties residing in an intrinsically closed state. We therefore propose that considerable differences between the respective eukaryote and prokaryote protein-conducting channel units and their regulation exist., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

SCY-635 is a novel nonimmunosuppressive cyclosporine-based analog that exhibits potent suppression of hepatitis C virus (HCV) replication in vitro. SCY-635 inhibited the peptidyl prolyl isomerase activity of cyclophilin A at nanomolar concentrations but showed no detectable inhibition of calcineurin phosphatase activity at concentrations up to 2 microM. Metabolic studies indicated that SCY-635 did not induce the major cytochrome P450 enzymes 1A2, 2B6, and 3A4. SCY-635 was a weak inhibitor and a poor substrate for P-glycoprotein. Functional assays with stimulated Jurkat cells and stimulated human peripheral blood mononuclear cells indicated that SCY-635 is a weaker inhibitor of interleukin-2 secretion than cyclosporine. A series of two-drug combination studies was performed in vitro. SCY-635 exhibited synergistic antiviral activity with alpha interferon 2b and additive antiviral activity with ribavirin. SCY-635 was shown to be orally bioavailable in multiple animal species and produced blood and liver concentrations of parent drug that exceeded the 50% effective dose determined in the bicistronic con1b-derived replicon assay. These results suggest that SCY-635 warrants further investigation as a novel therapeutic agent for the treatment of individuals who are chronically infected with HCV.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K(i) value of 3.8 mum. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.

Reversible and non-invasive photoswitching of the immunosuppressive effect of a drug would be a very valuable tool for precisely regulating the immune system. Using a combination of protein borrowing and two-photon photoisomerization, we designed and synthesized derivatives of cyclosporin A. Here we demonstrate photoswitching of the local conformation within small molecules, which we used to modulate inhibitory potencies for cyclophilin, influence ternary and quaternary complex formations and regulate T-cell transcriptional activation in situ.

Previous electrophysiological experiments characterized the Sec61 complex, which provides the aqueous path for entry of newly-synthesized polypeptides into the mammalian endoplasmic reticulum, as a highly dynamic channel that, once activated by precursor proteins, fluctuates between main open states with mean conductances of 220 and 550pS. Millimolar concentrations of lanthanum ions simultaneously restricted the dynamics of the Sec61 channel and inhibited translocation of polypeptides. Molecular modeling indicates that lanthanum binding sites cluster at the putative lateral gate of the Sec61 complex and suggests that structural flexibility of the lateral gate is essential for channel and protein transport activities of the Sec61 complex.

Three alcoholics (62 years, 47 years, 83 years) died between 11 and 18 h after ingestion of ethylene glycol (EG). One person committed suicide. Observed symptoms of intoxication were seizures, respiratory depression, arrhythmias and hypotonia. All died in hospital after failed attempts at resuscitation, one person did so after an 11h dialysis treatment. EG was detected in blood in concentrations of between 1 and 3mg/L (toxic range: >0.3mg/L). One case presented a blood alcohol concentration (bac) of 1.14 per thousand. Further toxic substances were not found. Using special staining techniques, oxalate crystals were found in samples from the kidneys, explaining renal failure, and in the medial layer of cerebral vessels.

A 65 year old woman had collected some plant leaves, cooked them in water and ate this mash with honey. A few hours after consumption first symptoms of intoxication like sickness, stomach aches and diarrhoea appeared. Her condition deteriorated, she was admitted to hospital, became comatose and finally died due to a multiorgan failure. The plants she had collected were identified as autumn crocus (Colchicum autumnale). Colchicine-concentrations detected in blood samples (HPLC/DAD) taken in hospital approximately 24 h after ingestion were 50 microg/l, in post-mortem femoral blood 65 microg/l colchicine were found (toxic range: >24 microg/l). Also all tested organs contained equally high colchicine levels. Histological examination of the liver showed diffuse vacuolization in the cytoplasma of hepatocytes. Possibly due to the relatively short survival time, only isolated mitotic structures were found within the epithelium of the colon.

Three different genes of catalytic subunit A of the Ca(2+)-dependent serine/threonine protein phosphatase calcineurin (CaN) are encoded in the human genome forming heterodimers with regulatory subunit B. Even though physiological roles of CaN have been investigated extensively, less is known about the specific functions of the different catalytic isoforms. In this study, all human CaN holoenzymes containing either the alpha, beta, or gamma isoform of the catalytic subunit (CaN alpha, beta, or gamma, respectively) were expressed for the first time. Comparative kinetic analysis of the dephosphorylation of five specific CaN substrates provided evidence that the distinct isoforms of the catalytic subunit confer substrate specificities to the holoenzymes. CaN alpha dephosphorylates the transcription factor Elk-1 with 7- and 2-fold higher catalytic efficiencies than the beta and gamma isoforms, respectively. CaN gamma exhibits the highest k(cat)/K(m) value for DARPP-32, whereas the catalytic efficiencies for the dephosphorylation of NFAT and RII peptide were 3- and 5-fold lower, respectively, when compared with the other isoforms. Elk-1 and NFAT reporter gene activity measurements revealed even more pronounced substrate preferences of CaNA isoforms. Moreover, kinetic analysis demonstrated that CaN beta exhibits for all tested protein substrates the lowest K(m) values. Enzymatic characterization of the CaN beta(P14G/P18G) variant as well as the N-terminal truncated form CaN beta(22-524) revealed that the proline-rich sequence of CaN beta is involved in substrate recognition. CaN beta(22-524) exhibits an at least 4-fold decreased substrate affinity and a 5-fold increased turnover number. Since this study demonstrates that all CaN isoforms display the same cytoplasmic subcellular distribution and are expressed in each tested cell line, differences in substrate specificities may determine specific physiological functions of the distinct isoforms.

FKBP36 has been previously shown to be a crucial factor in spermatogenesis because of its interplay with the synaptonemal complex protein SCPI. Here we show that beyond this function, FKBP36 forms complexes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) and Hsp90. Both proteins bind independently to different sites of the FKBP36 tetratricopeptide repeat domain. The interaction between FKBP36 and GAPDH directly inhibits the catalytic activity of GAPDH. In addition, FKBP36 expression causes a significant reduction of the GAPDH level and activity in COS-7 cells. Particularly in the cytosolic fraction, GAPDH was depleted by FKBP36 expression. Thus, FKBP36 diminishes GAPDH activity by direct interaction and down-regulation of GAPDH, which represents a previously unknown mechanism of GAPDH regulation and a novel function of FKBP36 in testis-specific signaling.

Ferrets (Mustela putorius furo) are becoming increasingly popular as pets and are kept in households with other pet animals such as dogs and cats, from which they may catch flea infestations with the predominant flea species Ctenocephalides felis. In this study, the efficacy of imidacloprid/moxidectin spot-on (Advocate/Advantage Multi) was investigated in the therapy and prevention of flea infestation in ferrets. Sixteen adult ferrets of varying weights and ages and of both sexes were included. Ferrets were infested with 50 fleas each on days -7, -1, 7, 14, 21, and 28. On study day -6, the animals were randomized into two groups based on body weight and flea count. Each ferret allocated to group 1 was treated with 0.4 ml of imidacloprid 10%/moxidectin 1% (Advocate/Advantage Multi). The ferrets in group 2 remained untreated. Flea counts were performed by combing 24 to 48 h after infestation. The primary efficacy criterion was the reduction in the number of fleas at each time point post treatment compared to the ferrets in the untreated control group. On day 1, the therapeutic efficacy was 100%. The preventative efficacy was 100% at 1 and 2 weeks post treatment, and it was >97% and >90% at 3 and 4 weeks post treatment. No local or systemic side effects were observed in any of the ferrets treated.

The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.

The peptidyl prolyl cis/trans isomerase (PPIase) Pin1 plays an important role in phosphorylation-dependent events of the cell cycle. This function is linked to its display of two phosphothreonine/phosphoserine-proline binding motifs, one within the type IV WW domain and a second within the parvulin-like catalytic domain. By microinjection of the compound Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2, which inhibits Xenopus laevis Pin1 with a Ki value of 19.4+/-1.5 nM, into the animal pole of X. laevis embryos at the two-cell stage, the impact of Pin1 PPIase activity on cell cycle progression and embryonic development could be analysed, independent of WW domain-mediated phosphoprotein binding. Injected embryos showed a dramatically decreased survival rate at late stages of development that could only be partially compensated by co-injection with mRNAs of enzymatically active Pin1 variants, demonstrating that the phosphorylation-specific PPIase activity of Pin1 is essential for cell division and development in X. laevis.

Dephosphorylation of NFAT by the Ca(2+)-calmodulin-dependent Ser/Thr protein phosphatase calcineurin is a bottleneck of T cell receptor-dependent activation of T cells. In dimeric complexes with immunophilins, the immunosuppressants cyclosporine A (CsA) and tacrolimus (FK506) block this process by inhibition of the enzymatic activity of calcineurin. We have identified the pyrazolopyrimidine compound NCI3 as a novel inhibitor of calcineurin-NFAT signaling. Similar to CsA and FK506, NCI3 inhibits dephosphorylation and nuclear translocation of NFAT, IL-2 production and proliferation of stimulated human primary T cells with IC(50) values from 2 to 4.5 microM. However, contrary to CsA and FK506, NCI3 neither blocks calcineurin;s phosphatase activity nor requires immunophilins for inhibiting NFAT activation. Our data suggest that NCI3 binds to calcineurin and causes an allosteric change interfering with NFAT dephosphorylation in vivo but not in vitro. NCI3 acts not only on the endogenous calcineurin but also on a C-terminally truncated, constitutively active version of calcineurin. The novel inhibitor described herein will be useful in better defining the cellular regulation of calcineurin activation and may serve as a lead for the development of a new type of immunosuppressants acting not by direct inhibition of the calcineurin phosphatase activity.

FKBP38 is a negative effector of the anti-apoptotic Bcl-2 protein in neuroblastoma cells. The interaction with Bcl-2 and the enzyme activity of FKBP38 depend on prior binding of calmodulin-Ca(2+) (CaM-Ca(2+)) at high Ca(2+) concentrations. The FKBP38 protein structure contains three tetratricopeptide repeat (TPR) motifs corresponding to the Hsp90 interaction sites of other immunophilins. In this study we show that the TPR domain of FKBP38 interacts with the C-terminal domain of Hsp90, but only if the FKBP38-CaM-Ca(2+) complex is preformed. Hence, FKBP38 is the first example of a TPR-containing immunophilin that interacts cofactor-dependently with Hsp90. In the ternary Hsp90-FKBP38-CaM-Ca(2+) complex the active site of FKBP38 is blocked, thus preventing interactions with Bcl-2. The dual control of the active site cleft of FKBP38 by CaM-Ca(2+) and Hsp90 highlights the importance of the enzyme activity of the FKBP38-CaM-Ca(2+) complex in the regulation of programmed cell death.

Multiple intracellular receptors of the FK506 binding protein (FKBP) family of peptidylprolyl cis/trans-isomerases are potential targets for the immunosuppressive drug FK506. Inhibition of the protein phosphatase calcineurin (CaN), which has been implicated in the FK506-mediated blockade of T cell proliferation, was shown to involve a gain of function in the FKBP12/FK506 complex. We studied the potential of six human FKBPs to contribute to CaN inhibition by comparative examination of inhibition constants of the respective FK506/FKBP complexes. Interestingly, these FKBPs form tight complexes with FK506, exhibiting comparable dissociation constants, but the resulting FK506/FKBP complexes differ greatly in their affinity for CaN, with IC50 values in the range of 0.047-17 microM. The different capacities of FK506/FKBP complexes to affect CaN activity are partially caused by substitutions corresponding to the amino acid side chains K34 and I90 of FKBP12. Only the FK506 complexes of FKBP12, FKBP12.6, and FKBP51 showed high affinity to CaN; small interfering RNA against these FKBP allowed defining the contribution of individual FKBP in an NFAT reporter gene assay. Our results allow quantitative correlation between FK506-mediated CaN effects and the abundance of the different FKBPs in the cell.

Immunoassays are presently used worldwide for the rapid screening of drugs. Despite the fact that they are a highly valuable tool for the testing of legal and illicit drugs, there is a real risk of false-positive and false-negative findings and many pitfalls must be taken into account when these tests are used in an uncritical manner and without valid confirmation procedures. In a long-standing successful cooperation with a large psychiatric hospital, we checked doubtful and nonplausible immunochemical findings in urine with gas chromatography-mass spectrometry (GC-MS) confirmation methods. The reported case histories demonstrate typical pitfalls, for example, influence of changing nutritional habits, use of sweeteners (cyclamate), uncritical creatinine correction, impaired elimination, or cross-reactivities of simultaneous therapeutic medication. In accordance with national and international associations, immunoassays are recommended as a useful tool for screening. However, confirmation analysis with conclusive methods (GC-MS or liquid chromatography-MS) is unavoidable for valid substance identification, discrimination between active and inactive metabolites, detection of congeners, and accurate determination of concentrations in body fluids.