Frequency and clinical impact of CDKN2A/ARF/CDKN2B gene deletions as assessed by in-depth genetic analyses in adult T cell acute lymphoblastic leukemia.

Recurrent deletions of the CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.

Diagnosis and New Treatment Modalities for Glioblastoma: Do They Improve Patient Survival?

Central nervous system (CNS) malignances include tumors of the brain and spinal cord. Taking into account the cell type where they originate from, there are almost 120 different types of CNS tumors. Benign tumors are not aggressive and normally do not invade other organs; however, they require surgical removal before they alter the surrounding brain functions. Primary malignant brain tumors commonly include astrocytomas, oligodendrogliomas, and ependimomas, where astrocytomas represent around 76%. The World Health Organization (WHO) has defined four histological grades of astrocytomas that range from the less aggressive tumors (grade I) to highly malignant tumors (grade IV). These grade IV tumors, also called glioblastoma (GBM), are the most aggressive of the primary malignant brain tumors. Patients with GBM have a median survival of 12 to 15 months. Current treatment for GBM includes surgery, radiotherapy and chemotherapy. Although there have been some advances in diagnosis and treatment, there is still no optimal treatment available for GBMs. In this review, we will discuss the approaches for GBM diagnosis and treatment, with a special emphasis to post-treatment imaging, and whether novel targeted therapies have impacted the survival of GBM patients. In addition, we will discuss clinical trials and the future of GBM diagnosis and treatment.

Inhibition of tryptophan hydroxylase abolishes fatigue induced by central tryptophan in exercising rats.

Fatigue during prolonged exercise is related to brain monoamines concentrations, but the mechanisms underlying this relationship have not been fully elucidated. We investigated the effects of increased central tryptophan (TRP) availability on physical performance and thermoregulation in running rats that were pretreated with parachlorophenylalanine (p-CPA), an inhibitor of the conversion of TRP to serotonin. On the 3 days before the experiment, adult male Wistar rats were treated with intraperitoneal (ip) injections of saline or p-CPA. On the day of the experiment, animals received intracerebroventricular (icv) injections of either saline or TRP (20.3 µM) and underwent a submaximal exercise test until fatigue. Icv TRP-treated rats that received ip saline presented higher heat storage rate and a 69% reduction in time to fatigue compared with the control animals. Pretreatment with ip p-CPA blocked the effects of TRP on thermoregulation and performance. Moreover, ip p-CPA administration accelerated cutaneous heat dissipation when compared with saline-pretreated rats. We conclude that an elevated availability of central TRP interferes with fatigue mechanisms of exercising rats. This response is modulated by serotonergic pathways, because TRP effects were blocked in the presence of p-CPA. Our data also support that a depletion of brain serotonin facilitates heat loss mechanisms during exercise.

Fatigue is mediated by cholinoceptors within the ventromedial hypothalamus independent of changes in core temperature.

We investigated brain mechanisms modulating fatigue during prolonged physical exercise in cold environments. In a first set of studies, each rat was subjected to three running trials in different ambient temperatures (T(a)). At 8 °C and 15 °C, core body temperature (T(core)) decreased and increased, respectively, whereas at 12 °C, the T(core) did not change throughout the exercise. In another set of experiments, rats were randomly assigned to receive bilateral 0.2 µL injections of 2.5 × 10(-2) M methylatropine or 0.15 M NaCl solution into the ventromedial hypothalamic nuclei (VMH). Immediately after the injections, treadmill exercise was started. Each animal was subjected to two experimental trials at one of the following T(a) : 5 °C, 12 °C or 15 °C. Muscarinic blockade of the VMH reduced the time to fatigue (TF) in cold environments by 35-37%. In all T(a) studied, methylatropine-treated rats did not present alterations in T(core) and tail skin temperature compared with controls. These results indicate that, below the zone of thermoneutrality, muscarinic blockade of the VMH decreases the TF, independent of changes in T(core). In conclusion, our data suggest that VMH muscarinic transmission modulates physical performance, even when the effects of thermoregulatory adjustments on fatigue are minimal.

Purification and characterization of a protein capable of binding to fatty acids and bile salts in Giardia lamblia.

A specific fatty acid binding protein was isolated from Giardia lamblia, using an affinity column with butyric acid acting as a ligand in place of stearic acid. This method has proved to be more efficient than the one previously described using stearic acid as ligand. The purified fraction showed 8 electrophoretic bands of proteins, with molecular weights ranging between 8 and 80 kDa. This pattern is a consequence of the aggregation of a protein with a molecular weight of 8,215 Da, corresponding to the lower molecular weight band, the only one capable of binding to fatty acids. The labeled oleic acid bound to these purified proteins was replaced by a 100-fold greater concentration of taurocholate, glycocholate, deoxycholate, palmitic acid, and arachidonic acid, having a greater displacement of the bile salts than the free fatty acids.

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Physical exercise-induced cardiovascular adjustments are modulated by muscarinic cholinoceptors within the ventromedial hypothalamic nucleus.

The effects of blocking ventromedial hypothalamic nucleus (VMH) muscarinic cholinoceptors on cardiovascular responses were investigated in running rats. Animals were anesthetized with pentobarbital sodium and fitted with bilateral cannulae into the VMH. After recovering from surgery, the rats were familiarized to running on a treadmill. The animals then had a polyethylene catheter implanted into the left carotid artery to measure blood pressure. Tail skin temperature (T(tail)), heart rate, and systolic, diastolic and mean arterial pressure were measured after bilateral injections of 0.2 microl of 5 x 10(-9) mol methylatropine or 0.15 M NaCl solution into the hypothalamus. Cholinergic blockade of the VMH reduced time to fatigue by 31 % and modified the temporal profile of cardiovascular and T(tail) adjustments without altering their maximal responses. Mean arterial pressure peak was achieved earlier in methylatropine-treated rats, which also showed a 2-min delay in induction of tail skin vasodilation, suggesting a higher sympathetic tonus to peripheral vessels. In conclusion, muscarinic cholinoceptors within the VMH are involved in a neuronal pathway that controls exercise-induced cardiovascular adjustments. Furthermore, blocking of cholinergic transmission increases sympathetic outflow during the initial minutes of exercise, and this higher sympathetic activity may be responsible for the decreased performance.

CTLA-4 genotype and relapse incidence in patients with acute myeloid leukemia in first complete remission after induction chemotherapy.

The recently described single-nucleotide polymorphism CT60, located in the 3'-untranslated region of the CTLA4 (cytotoxic T-lymphocyte antigen 4 ) gene, has been associated with susceptibility to several autoimmune diseases and has also been shown to be involved in immune responses following allogeneic stem cell transplantation (SCT). However, the contribution of the CTLA4 genotype to the control of minimal residual disease in patients with acute myeloid leukemia (AML) has yet to be explored. We investigated the association between the CTLA4 CT60 A/G genotype and the incidence of leukemic relapse in 143 adult patients with AML in first complete remission after the same chemotherapy protocol (CETLAM LAM'03). The CT60 AA genotype was associated with a higher rate of leukemic relapse (56.4 vs 35.6%, P=0.004; hazard ratio (HR)=2.64, 95% confidence interval (CI)=1.36-5.14) and lower overall survival at 3 years (39.4 vs 68.4%, P=0.004; HR=2.80, 95% CI=1.39-5.64). This is the first study to report an association between polymorphisms at CTLA-4 and AML relapse.

Intracerebroventricular physostigmine enhances blood pressure and heat loss in running rats.

The aim of this study was to evaluate the effects of the stimulation of central cholinergic synapses in the regulation of heat loss in untrained rats during exercise. The animals were separated into two groups (exercise or rest) and tail skin temperature (T(tail)), core temperature and blood pressure were measured after injection of 2 microL of 5x10(-3) M physostigmine (Phy; n = 8) or 0.15 M NaCl solution (Sal; n = 8) into the lateral cerebral ventricle. Blood pressure was recorded by a catheter implanted into the abdominal aorta, T(tail) was measured using a thermistor taped to the tail and intraperitoneal temperature (T(b)) was recorded by telemetry. During exercise, Phy-treated rats had a higher increase in mean blood pressure (147 +/- 4 mmHg Phy vs. 121 +/- 3 mmHg Sal; P < 0.001) and higher T(tail) (26.4 +/- 1.0 degrees C Phy vs. 23.8 +/- 0.5 degrees C Sal; P < 0.05) that was closely related to the increase in systolic arterial pressure (r = 0.83; P < 0.001). In addition, Phy injection attenuated the exercise-induced increase in T(b) compared with controls without affecting running time. We conclude that the activation of central cholinergic synapses during exercise increases heat dissipation due to the higher increase in blood pressure.

Characterization of the satellite DNA Msat-160 from the species Chionomys nivalis (Rodentia, Arvicolinae).

The satellite DNA Msat-160 has been previously characterized in several species of the genus Microtus. Here we present the characterization of Msat-160 from Chionomys nivalis, a species with a very primitive karyotype. As in other Microtus species analyzed, C. nivalis Msat-160 is AT rich, has a monomer length of 160 bp, is undermethylated and is mainly located in all the pericentromeric heterochromatin of all autosomes and the X chromosome, but is completely absent from the Y chromosome. Hence, our results support the hypothesis that Msat-160 was initially distributed in the pericentromeric heterochromatin of all autosomes and the X chromosome. The taxonomic status of the genus Chionomys in relation to the genus Microtus is a very interesting issue, so we constructed phylogenetic dendrograms using Msat-160 sequences from several Microtus species. Although the results were not informative about this issue, the presence of Msat-160 in C. nivalis and Microtus species suggested that both genera are closely related and that this satellite DNA was present in the common ancestor. Studies of Msat-160 in different arvicoline species could help to determine the origin of this satellite and, perhaps, to establish the phylogenetic relationships of some arvicoline groups.

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications.

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the 'selfish DNA' hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged ('giant') sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.

Binding properties and immunolocalization of a fatty acid-binding protein in Giardia lamblia.

We describe here a fatty acid-binding protein (FABP) isolated and purified from the parasitic protozoon Giardia lamblia. The protein has a molecular mass of 8 kDa and an isoelectric point of 4.96. A Scatchard analysis of the data at equilibrium revealed a dissociation constant of 3.12 x 10(-8) M when the labeled oleic acid was displaced by a 10-fold greater concentration of unlabeled oleic acid. Testosterone, sodium desoxycholate, taurocholate, metronidazol, and alpha-tocopherol, together with butyric, arachidonic, palmitic, retinoic, and glycocholic acids, were also bound to the protein. Assays with polyclonal antibodies revealed that the protein is located in the ventral disk and also appears in the dorsal membrane, the cytoplasm, and in the vicinity of the lipid vacuoles.

Host humoral immune response to Leishmania lipid-binding protein.

SUMMARY We report on the use of Leishmania donovani lipid-binding proteins (LBPs) as antigens capable of being recognized by serum from immunocompetent patients from southern Spain suffering from visceral leishmaniasis and from Peruvian patients with localized cutaneous leishmaniasis caused by Leishmania braziliensis. The absorbance found by immunoenzymatic techniques gave significantly different results for the serum samples from patients with and without leishmaniasis. Specificity by ELISA testing was 93.2% and sensibility 100%. Dot blots from human patient serum samples or naturally infected dogs from Spain gave similarly significant results. All the human serum samples from individuals with visceral leishmaniasis and the Leishmania-positive canine samples recognized two bands, with molecular weights of 8 and 57 kDa. The serum from individuals with cutaneous leishmaniasis caused by L. braziliensis recognized an additional band of 16 kDa. We discuss the role of Leishmania FABP and compare the immunological reactions found with serum samples from other protozoan infections such as toxoplasma and Chagas as well as bacterial infections such as tuberculosis and syphilis.

Characterization of an EcoRI family of satellite DNA from two species of the genus Eptesicus (Vespertilionidae; Chiroptera).

We have cloned and sequenced a 321 bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321 bp which is composed of two subunits of 160 bp, suggested by the existence of a 160 bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.

Transmission analysis of B chromosomes in Rattus rattus from Northern Africa.

Traditionally, B chromosomes have been classified as parasitic or heterotic, depending of whether or not they show selfish behaviour. Nevertheless, experimental evidence has been found supporting the idea that supernumerary chromosomes may evolve from parasitism to neutrality. In this work, B chromosome transmission in Rattus rattus has been analysed by performing several crosses between individuals carrying different numbers of supernumerary chromosomes. Our results demonstrated a Mendelian transmission rate through males, but slight accumulation of the Bs through females. This parasitic behaviour is shared in populations as distant as Asia and Africa, and even in a related species in Australia, suggesting the possibility of an ancient origin of these supernumerary chromosomes.

X chromosome painting in Microtus: origin and evolution of the giant sex chromosomes.

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as 'giant sex chromosomes') due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.

A repeat DNA sequence from the Y chromosome in species of the genus Microtus.

In most mammals, the Y chromosome is composed of a large amount of constitutive heterochromatin. In some Microtus species, this feature is also extended to the X chromosome, resulting in enlarged (giant) sex chromosomes. Several repeated DNA sequences have been described in the gonosomal heterochromatin of these species, indicating that it has heterogeneous and species-specific composition and distribution. We have cloned an AT-rich, 851-bp long, repeated DNA sequence specific for M. cabrerae Y chromosome heterochromatin. The analysis of other species of the genus Microtus indicated that this sequence is also located on the Y chromosome (male-specific) in three species (M. agrestis, M. oeconomus and M. nivalis), present on both Y and X chromosomes and on some autosomes in M. arvalis and absent in the genome of M. guentheri. Our data also suggest that the mechanism of heterochromatin amplification operating on the sex chromosomes could have been different in each species since the repeated sequences of the gonosomal heterochromatic blocks in M. cabrerae and M. agrestis are different. The absence of this sequence in the mouse genome indicates that its evolutionary origin could be recent. Future analysis of the species distribution, localization and sequence of this repeat DNA family in arvicolid rodent species could help to establish the unsolved phylogenetic relationships in this rodent group.

Sex chromosomes, sex determination, and sex-linked sequences in Microtidae.

The Arvicolidae is a widely distributed rodent group with several interesting characteristics in their sex chromosomes. Here, we summarize the actual knowledge of some of these characteristics. This mammalian group has species with abnormal sex determination systems. In fact, some species present the same karyotype in both males and females, with total absence of a Y chromosome, and hence of SRY and ZFY genes. Other species present fertile, sex-reversed XY females, generally due to mutations affecting X chromosomes. Furthermore, in Microtus oregoni males and females are gonosomic mosaic (the females are XO in the soma and XX in the germ cells, while the males are XY in the soma and OY in the germ cells). Regarding sex chromosomes, some species present enlarged (giant) sex chromosomes because of the presence of large blocks of constitutive heterochromatin, which have been demonstrated to be highly heterogeneous. Furthermore, we also consider the alterations affecting composition and localization of sex-linked genes or repeated sequences. Finally, this rodent group includes species with synaptic and asynaptic sex chromosomes. In fact, several species with asynaptic sex chromosomes have been described. It is interesting to note that within the genus Microtus both types of sex chromosomes are present.

Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera).

This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.