Screening for gene mutations in a 500 kb neuroblastoma tumor suppressor candidate region in chromosome 1p; mutation and stage-specific expression in UBE4B/UFD2

Deletion of a part of the short arm of chromosome 1 is one of the most common chromosomal rearrangements observed in neuroblastoma (NBL) tumors and it is associated with a poor prognosis. No NBL tumor suppressor gene has yet been identified in the region. Our shortest region of overlap of deletions, ranging from marker D1S80 to D1S244, was shown to partly overlap a 500 kb region that was homozygously deleted in a NBL cell line. We have screened seven genes known to reside in or very close to this overlap consensus region, UBE4B/UFD2, KIF1B, DFFA, PGD, CORT, PEX14, and ICAT, for coding mutations in NBL tumor DNA. A few deviations from the reference sequences were identified; most interestingly being a splice site mutation that was detected in UBE4B/UFD2 in a stage 3 NBL with a fatal outcome. This mutation was neither present in the patients constitutional DNA nor in any of 192 control chromosomes analysed. Also, the expression of UBE4B/UFD2 was markedly diminished in the high-stage/poor-outcome tumors as compared to the low-stage/favorable-outcome tumors. Overall, the number of amino-acid changes in the genes of the region was low, which shows that mutations in these genes are rare events in NBL development. Given the data presented here, UBE4B/UFD2 stands out as the strongest candidate NBL tumor suppressor gene in the region at this stage.     

Stress-induced hyperthermia and anxiety: pharmacological validation

When mammals, including man, are confronted with a stressful event, their core body temperature rises, stress-induced hyperthermia. In mice, the stress-induced hyperthermia procedure has been developed to measure antistress or anxiolytic-like effects of psychoactive drugs. Group-housed and singly housed versions of the stress-induced hyperthermia generate comparable results. Because the number of animals needed to perform an experiment is much lower in the singly housed versus the group-housed procedure, the former is the test of choice for pharmacological testing. A typical stress-induced hyperthermia test starts with an injection 60 min before the first rectal temperature measurement (T(1)), followed by a second temperature measurement (T(2)) 10-15 min later. The difference DeltaT (=T(2)-T(1)) is the stress-induced hyperthermia. The procedure also measures the intrinsic activity of drugs on the basal body temperature and DeltaT is relatively independent from the intrinsic temperature effects of drugs. Anxiolytic drugs (benzodiazepines, 5-HT(1A) receptor agonists, alcohol) reduce DeltaT suggestive of anxiolytic-like effects. Because the parameter measured for anxiety in the stress-induced hyperthermia procedure is not dependent on locomotor activity, like in almost all other anxiety tests, the stress-induced hyperthermia procedure is an attractive addition to tests in the anxiety field. Because the stress-induced hyperthermia is also present with a comparable pharmacological profile in females, this procedure has a wide species and gender validity. The procedure was applied in various genetically modified mice [5-HT(1A) and 5-HT(1B) receptor knockout (KO) mice and corticotropin-releasing hormone overexpressing (CRH-OE) mice] to study phenotypic influences of the various mutations on aspects of anxiety. The stress-induced hyperthermia test in singly housed male and female mice appears a useful and extremely simple test to measure effects of drugs on certain aspects of anxiety or to help to determine phenotypic differences in mutant mice.     

Identification of human cytochrome P(450)s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data

Objective Knowledge about the metabolism of anti-parasitic drugs (APDs) will be helpful in ongoing efforts to optimise dosage recommendations in clinical practise. This study was performed to further identify the cytochrome P₄₅₀ (CYP) enzymes that metabolise major APDs and evaluate the possibility of predicting in vivo drug clearances from in vitro data.Methods In vitro systems, rat and human liver microsomes (RLM, HLM) and recombinant cytochrome P₄₅₀ (rCYP), were used to determine the intrinsic clearance (CL_int) and identify responsible CYPs and their relative contribution in the metabolism of 15 commonly used APDs.Results and discussion CL_int determined in RLM and HLM showed low (r²=0.50) but significant (P<0.01) correlation. The CL_int values were scaled to predict in vivo hepatic clearance (CL_H) using the 'venous equilibrium model'. The number of compounds with in vivo human CL data after intravenous administration was low (n=8), and the range of CL values covered by these compounds was not appropriate for a reasonable quantitative in vitro–in vivo correlation analysis. Using the CL_H predicted from the in vitro data, the compounds could be classified into three different categories: high-clearance drugs (>70% liver blood flow; amodiaquine, praziquantel, albendazole, thiabendazole), low-clearance drugs (<30% liver blood flow; chloroquine, dapsone, diethylcarbamazine, pentamidine, primaquine, pyrantel, pyrimethamine, tinidazole) and intermediate clearance drugs (artemisinin, artesunate, quinine). With the exception of artemisinin, which is a high clearance drug in vivo, all other compounds were classified using in vitro data in agreement with in vivo observations. We identified hepatic CYP enzymes responsible for metabolism of some compounds (praziquantel—1A2, 2C19, 3A4; primaquine—1A2, 3A4; chloroquine—2C8, 2D6, 3A4; artesunate—2A6; pyrantel—2D6). For the other compounds, we confirmed the role of previously reported CYPs for their metabolism and identified other CYPs involved which had not been reported before.Conclusion Our results show that it is possible to make in vitro–in vivo predictions of high, intermediate and low CL_int drug categories. The identified CYPs for some of the drugs provide a basis for how these drugs are expected to behave pharmacokinetically and help in predicting drug–drug interactions in vivo.     

High leptin levels are associated with stroke

BACKGROUND AND PURPOSE: Leptin, an important hormone for body weight regulation, may be involved in the pathogenesis of cardiovascular manifestations of obesity. We tested whether leptin may be an independent risk marker for stroke in a case-referent study. METHODS: Definitive acute stroke events, defined by MONICA criteria, were identified from October 1, 1995 to April 30, 1999. Referents without known cardiovascular disease were randomly selected from a population census. Patient characteristics were taken from hospital files and leptin was analyzed in stored samples. Logistic regression analysis was used to determine possible differences in leptin levels between groups. RESULTS: One hundred and thirty-seven cases with ischemic stroke and 69 cases with hemorrhagic stroke were identified. In comparison with referents, male patients with stroke had significantly higher leptin levels. Both male and female stroke patients had increased blood pressure compared with the referents. In multivariate analyses, high leptin levels were associated with both ischemic (OR = 4.89; 95% CI: 1.89-12.62) and hemorrhagic (OR = 3.86; 95% CI: 1.13-13.16) stroke in men, and with ischemic stroke in women (OR = 4.10; 95% CI: 1.45-11.62). The combination of high leptin levels and increased blood pressure (systolic or diastolic) was associated with a strong positive interaction in males with hemorrhagic stroke. CONCLUSION: Leptin may be an important link for the development of cerebrovascular disease in the insulin resistance syndrome in men.     

Pituitary adenylate cyclase-activating polypeptide prevents C2-ceramide-induced apoptosis of cerebellar granule cells

The sphingolipid metabolites, ceramides, are critical mediators of the cellular stress response and play an important role in the control of programmed cell death. In particular, ceramides have been shown to induce apoptosis of cerebellar granule cells. We show that pituitary adenylate cyclase-activating polypeptide (PACAP) prevents C2-ceramide-induced apoptosis. The neuroprotective effect of PACAP was dose-dependent and blocked by its antagonist, PACAP6-38, whereas the PACAP-related peptide VIP was inactive. The effect of PACAP on cell survival was mimicked by dibutyryl-cAMP (dbcAMP) and forskolin and prevented by the MEK inhibitor U0126, indicating that both the adenylyl-cyclase and MAP-kinase pathways contribute to the neuroprotective action of the peptide. C2-ceramide and PACAP induced opposite effects on phosphorylated forms of ERK and JNK without affecting the total amounts of ERK and JNK, suggesting that a balance between these two MAP-kinases is critical for the cell survival/death decision. The effect of PACAP on ERK phosphorylation was blocked by U0126, but was not affected by H89 or chelerythrine indicating that PACAP activates ERK through a PKA- and PKC-independent mechanism. C2-ceramide induced a time-dependent activation of caspase-3, enhanced the amount of cleaved caspase-3 and stimulated the DNA fragmentation process, while PACAP strongly inhibited the C2-ceramide-induced activation of caspase-3, reduced the expression of cleaved caspase-3 and blocked DNA fragmentation. Taken together, the present results show that C2-ceramide induces apoptosis of cerebellar granule cells through a mechanism involving activation of caspase-3. Our data also demonstrate that PACAP is a potent inhibitor of C2-ceramide-induced apoptosis.     

Are factors associated with subjective quality of life in people with severe mental illness consistent over time? — A 6-year follow-up study

Objective To investigate the cross-sectional relationship between subjective quality of life and sociodemographic clinical and social factors over three points of assessment during a 6-year follow-up, and to investigate longitudinal predictors of subjective quality of life. Method We investigated a sample of people with severe mental illness (n = 92), mainly with a psychosis diagnosis, at baseline and at an 18-month and 6-year follow-up. Measures included the Lancashire quality of life profile, Manchester short assessment of quality of life, Symptom Check List 90, Camberwell Assessment of Needs and the Interview Schedule for Social Interaction. Results Cross-sectionally subjective quality of life was associated to self-reported symptoms, social network and unmet needs. However, these determinants varied in importance between points of assessment. Longitudinal predictors of subjective quality of life were changes in self-reported symptoms and social network. Conclusion There was a rather consistent set of determinants of subjective quality of life over time. Social network seems to be an important factor with relevance for improvements in subjective quality of life, however largely overlooked in earlier studies within the field.     

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15–18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM. Received: 13 July 1999 / Revised: 6 October 1999 · Accepted: 12 October 1999