Effect of testosterone metabolites on ABC half-transporter relative gene expression in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with reduced very long-chain fatty acid beta-oxidation, mainly affecting the nervous system, the adrenal cortex and the testes. The clinical manifestations of hypogonadism, alopecia and the impairment of the enzyme 5alpha-reductase, which converts testosterone into dihydrotestosterone, clearly point to an involvement of androgens in this pathology. The disease is characterized by mutations in the ABCD1 gene, which codes for the peroxisomal ABC half-transporter ALDP, and by a broad range of clinical manifestations. The altered function of ALDP can be compensated by the overexpression of proteins belonging to the same family of ABC half-transporters. A promising therapeutic approach is represented by the activation of these proteins by specific agonists. In this study we evaluated the effect of the testosterone metabolite dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) on the expression of the ABC half-transporters encoded by the ABCD2 and ABCD3 genes, in fibroblasts drawn from controls and from two affected brothers. The two patients presented the same mutation in exon 9 but had different clinical manifestations, one patient being asymptomatic and the second one severely affected. When the cells were stimulated with testosterone metabolites, only the severely affected patient showed a significant increase in ABCD2 mRNA levels, while the ABCD3 expression remained unchanged in both patients

Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro

Little is known about the interaction of novel anticancer drugs with other treatment modalities. The aim of this study was to examine the effect of combining imatinib mesylate (STI-571) with radiation or cisplatin on the survival of two human solid tumor cell lines – SKNMC cells derived from Ewing sarcoma and breast cancer MCF-7 cells. Methods: Cell proliferation was determined using the sulphorodamine B cytotoxicity assay. Cell cycle analysis was performed with flow cytometry. Apoptosis was determined using a commercial cell death ELISA plus kit. Phosphorylated AKT, which has been suggested to be involved in radiation resistance, was detected by Western blot analysis. Results: Exposure of SKNMC cells to STI-571 resulted in a dose-dependent antiproliferative effect and a decrease in phosphorylated AKT expression. There was no evidence of apoptosis. The combination of STI-571 with radiation or cisplatin had an additive antiproliferative effect in SKNMC cells (60% reduction in cell number). A similar effect was observed in human MCF-7 breast cancer cells. Conclusion: STI-571 improves the outcome of cisplatin or irradiation treatment in vitro. AKT pathway may play a role in the additive effect of STI-571 and irradiation.Цель: оценить антипролиферативный эффект иматиниба (STI-571) в комбинации с облучением или цисплатиной по отношению к двум клеточным линиям – клеткам линии SKNMC, полученным из саркомы Эвинга, и клеткам рака молочной железы человека линии MCF-7. Методы: для оценки пролиферации клеток применяли метод анализа цитотоксичности с использованием сульфородамина B. Для анализа распределения клеток по фазам клеточного цикла применяли метод проточной цитометрии, апоптоза – с применением коммерческого набора для проведения ИФА. Уровень фосфорилированной киназы АКТ, предположительно связанной с радиорезистентностью, определяли методом Вестерн-блот анализа. Результаты: инкубация клеток SKNMC STI-571 приводила к дозозависимому антипролиферативному эффекту и снижению фосфорилирования AKT, но не апоптозу клеток. Комбинированное применение STI-571 и обления или цисплатины оказывало дополнительное антипролиферативное воздействие на клетки линии SKNMC (60% уменьшения количества клеток). Аналогичные эффекты отмечали на клетках линии MCF-7. Выводы: обработка опухолевых клеток STI-571 усиливает эффект обления и цисплатины in, причем таковой может быть опосредован сигнальным каскадом AK

Increased serum levels of TGFβ1 in children with localized scleroderma

scleroderm

Anisotropic flow at RHIC: How unique is the number-of-constituent-quark scaling?

The transverse momentum dependence of the anisotropic flow $v_2$ for $\pi$, $K$, nucleon, $\Lambda$, $\Xi$ and $\Omega$ is studied for Au+Au collisions at $\sqrt{s_{\rm NN}} = 200$ GeV within two independent string-hadron transport approaches (RQMD and UrQMD). Although both models reach only 60% of the absolute magnitude of the measured $v_2$, they both predict the particle type dependence of $v_2$, as observed by the RHIC experiments: $v_2$ exhibits a hadron-mass hierarchy (HMH) in the low $p_T$ region and a number-of-constituent-quark (NCQ) dependence in the intermediate $p_T$ region. The failure of the hadronic models to reproduce the absolute magnitude of the observed $v_2$ indicates that transport calculations of heavy ion collisions at RHIC must incorporate interactions among quarks and gluons in the early, hot and dense phase. The presence of an NCQ scaling in the string-hadron model results suggests that the particle-type dependencies observed in heavy-ion collisions at intermediate $p_T$ might be related to the hadronic cross sections in vacuum rather than to the hadronization process itself.Comment: 10 pages, 5 figures; A new author (H. Petersen) is added; A new figure (fig.1) on time evolution of elliptic flow and number of collisions is added; Version accepted for publication in J. Phys.

Preferential expression of mutant ABCD1 allele is common in adrenoleukodystrophy female carriers but unrelated to clinical symptoms

<p>Abstract</p> <p>Background</p> <p>Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant <it>ABCD1 </it>allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.</p> <p>Methods</p> <p>To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify <it>ABCD1 </it>allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.</p> <p>Results</p> <p>We found severely (≥90:10) or moderately (≥75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant <it>ABCD1 </it>allele, irrespective of the manifestation of symptoms. The expression of mutant <it>ABCD1 </it>allele also correlates with plasma VLCFA concentrations.</p> <p>Conclusions</p> <p>Our results indicate that preferential XCI leads to the favored expression of the mutant <it>ABCD1 </it>allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant <it>ABCD1 </it>allele, but argue against the use of XCI pattern, <it>ABCD1 </it>allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.</p

The miR-17 similar to 92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c(-/-); Ptch1(+/-) and Ink4c(-/-); p53(-/-). We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17 similar to 92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17 similar to 92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17 similar to 92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c(-/-); Ptch1(+/-) mice. These, but not similarly engineered cells from Ink4c(-/-); p53(-/-) mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17 similar to 92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17 similar to 92 cluster and the SHH signaling pathway in the development of MBs in mouse and man

Genetic Background of Prop1df Mutants Provides Remarkable Protection Against Hypothyroidism-Induced Hearing Impairment

Hypothyroidism is a cause of genetic and environmentally induced deafness. The sensitivity of cochlear development and function to thyroid hormone (TH) mandates understanding TH action in this sensory organ. Prop1df and Pou1f1dw mutant mice carry mutations in different pituitary transcription factors, each resulting in pituitary thyrotropin deficiency. Despite the same lack of detectable serum TH, these mutants have very different hearing abilities: Prop1df mutants are mildly affected, while Pou1f1dw mutants are completely deaf. Genetic studies show that this difference is attributable to the genetic backgrounds. Using embryo transfer, we discovered that factors intrinsic to the fetus are the major contributor to this difference, not maternal effects. We analyzed Prop1df mutants to identify processes in cochlear development that are disrupted in other hypothyroid animal models but protected in Prop1df mutants by the genetic background. The development of outer hair cell (OHC) function is delayed, but Prestin and KCNQ4 immunostaining appear normal in mature Prop1df mutants. The endocochlear potential and KCNJ10 immunostaining in the stria vascularis are indistinguishable from wild type, and no differences in neurofilament or synaptophysin staining are evident in Prop1df mutants. The synaptic vesicle protein otoferlin normally shifts expression from OHC to IHC as temporary afferent fibers beneath the OHC regress postnatally. Prop1df mutants exhibit persistent, abnormal expression of otoferlin in apical OHC, suggesting delayed maturation of synaptic function. Thus, the genetic background of Prop1df mutants is remarkably protective for most functions affected in other hypothyroid mice. The Prop1df mutant is an attractive model for identifying the genes that protect against deafness