Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.     

A novel method for study of protein folding kinetics by monitoring diffusion coefficient in time domain

Molecular diffusion process after the photo-induced electron injection to ferric cytochrome c (Fe(III) cyt c) in guanidine hydrochloride (GdnHCl) 3.5 M buffer solution is studied by the time-resolved transient grating technique. Circular dichroism studies have revealed that Fe(III) cyt c is unfolded under this condition but the reduced form, Fe(II) cyt c, is folded. Hence, this pulsed laser-induced reduction should initiate the folding process of cyt c. The observed transient grating signal shows prominent features, which have never been observed before. Based on several characteristic points, we concluded that the apparent diffusion coefficient (D) of Fe(II) cyt c after the reduction is time dependent, which must be associated with the protein folding dynamics. This time-dependent apparent D should reflect either the continuous time development of the hydrodynamic radius or population change of the unfolded and folded states during the folding dynamics. This is the first observation of the time-dependent apparent D during any chemical reaction, and this time-dependent measurement of D should be a unique and powerful way to study the protein folding kinetics from a viewpoint of the protein's shape or the protein-water intermolecular interaction.     

Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit

Summary The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with -MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla.From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

ZO-1- and ZO-2-dependent integration of myosin-2 to epithelial zonula adherens

For the zonula adherens (ZA) to be established by linear arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here, we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 (zonula occludens-1 and -2) by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ (PSD-95/discs large/zonula occludens-1)-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2–dependent way to establish ZA from primordial forms in epithelial cells.     

Off-pump coronary bypass surgery adversely affects alveolar gas exchange

While the introduction of off-pump myocardial revascularization (OPCAB) has initially shown promise in reducing respiratory complications inherent to conventional coronary surgery, it has failed to eradicate them. Our study focused on quantifying the lactate release from the lungs and the dysfunction at the level of the alveolar-capillary membrane precipitated by OPCAB at different time points after the insult. Furthermore, we aimed to determine the impact of pulmonary lactate production on systemic lactic acid concentrations. The study was conducted in a prospective observational fashion. Forty consecutive patients undergoing OPCAB were analyzed. The mean patient age was 60 +/- 10 years. The mean EUROScore was 3.8 +/- 2.9. The alveolar-arterial O2 gradient increased from 19 [range 9 to 30] to 26 [range 20 to 34] kPa (P < 0.001) and remained elevated up to 6 hours after surgery. It rapidly declined again by 18 hours postoperatively. The observed increase in the pulmonary lactate release (PLR) from a baseline value of 0.022 [range -0.074 to 0.066] to 0.089 [range 0.016 to 0.209] mmol/min/m2 at six hours postoperatively did not reach statistical significance (P = 0.105). The systemic arterial lactate (Ls) concentration increased from 0.94 [range 0.78 to 1.06] to 1.39 [range 0.97 to 2.81] mmol/L (P < 0.001). The venoarterial pCO2 difference showed no significant change in comparison to baseline values. The mortality in the studied group was 2.5% (1/40). The pulmonary lactate production showed a statistically significant correlation with the systemic lactate concentration (R = 0.46; P = 0.003). Pulmonary injury following off pump myocardial revascularization was evidenced by a prompt increase in the alveolar-arterial oxygen gradient. The alveolar-arterial O2 gradient correlated with the duration of mechanical ventilation.     

Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

Deletion of Running-Induced Hippocampal Neurogenesis by Irradiation Prevents Development of an Anxious Phenotype in Mice

Recent evidence postulates a role of hippocampal neurogenesis in anxiety behavior. Here we report that elevated levels of neurogenesis elicit increased anxiety in rodents. Mice performing voluntary wheel running displayed both highly elevated levels of neurogenesis and increased anxiety in three different anxiety-like paradigms: the open field, elevated O-maze, and dark-light box. Reducing neurogenesis by focalized irradiation of the hippocampus abolished this exercise-induced increase of anxiety, suggesting a direct implication of hippocampal neurogenesis in this phenotype. On the other hand, irradiated mice explored less frequently the lit compartment of the dark-light box test irrespective of wheel running, suggesting that irradiation per se induced anxiety as well. Thus, our data suggest that intermediate levels of neurogenesis are related to the lowest levels of anxiety. Moreover, using c-Fos immunocytochemistry as cellular activity marker, we observed significantly different induction patterns between runners and sedentary controls when exposed to a strong anxiogenic stimulus. Again, this effect was altered by irradiation. In contrast, the well-known induction of brain-derived neurotrophic factor (BDNF) by voluntary exercise was not disrupted by focal irradiation, indicating that hippocampal BDNF levels were not correlated with anxiety under our experimental conditions. In summary, our data demonstrate to our knowledge for the first time that increased neurogenesis has a causative implication in the induction of anxiety.