Cytogenetics and molecular genetics of acute lymphoblastic leukemia

An important factor in the diagnosis of acute lymphoblastic leukemia (ALL) is that karyotype is an independent prognostic indicator, with an impact on the choice of treatment. Outcome is related to the number of chromosomes. For example, high hyperdiploidy (51-65 chromosomes) is associated with a good prognosis, whereas patients with near haploidy (23-29 chromosomes) have a poor outcome. The discovery of recurring chromosomal abnormalities in the leukemic blasts of patients with ALL has identified a large number of genes involved in leukemogenesis. Certain specific genetic changes are related to prognosis. The ETV6/AML1 fusion arising from the translocation (t12;21) (p13;q22) has been associated with a good outcome; the BCR/ABL fusion of (t9;22)(q34;q11), rearrangements of the MLL gene, and abnormalities of the short arm of chromosomes 9 involving the tumor suppressor genes p16INK4A have a poor prognosis. Unfortunately, the classification of patients into prognostic groups based on cytogenetics is not always as predicted. Even when other clinically based risk factors are taken into account, some patients with good-risk cytogenetic features will relapse. In the search for new measures of prognosis, it has recently emerged that the level of minimal residual disease following induction therapy can be a reliable predictor of outcome in ALL.     

Improved single- and multi-contact life-time testing of dental restorative materials using key characteristics of the human masticatory system and a force/position-controlled robotic dental wear simulator

This paper presents a new in vitro wear simulator based on spatial parallel kinematics and a biologically inspired implicit force/position hybrid controller to replicate chewing movements and dental wear formations on dental components, such as crowns, bridges or a full set of teeth. The human mandible, guided by passive structures such as posterior teeth and the two temporomandibular joints, moves with up to 6 degrees of freedom (DOF) in Cartesian space. The currently available wear simulators lack the ability to perform these chewing movements. In many cases, their lack of sufficient DOF enables them only to replicate the sliding motion of a single occlusal contact point by neglecting rotational movements and the motion along one Cartesian axis. The motion and forces of more than one occlusal contact points cannot accurately be replicated by these instruments. Furthermore, the majority of wear simulators are unable to control simultaneously the main wear-affecting parameters, considering abrasive mechanical wear, which are the occlusal sliding motion and bite forces in the constraint contact phase of the human chewing cycle. It has been shown that such discrepancies between the true in vivo and the simulated in vitro condition influence the outcome and the quality of wear studies. This can be improved by implementing biological features of the human masticatory system such as tooth compliance realized through the passive action of the periodontal ligament and active bite force control realized though the central nervous system using feedback from periodontal preceptors. The simulator described in this paper can be used for single- and multi-occlusal contact testing due to its kinematics and ability to exactly replicate human translational and rotational mandibular movements with up to 6 DOF without neglecting movements along or around the three Cartesian axes. Recorded human mandibular motion and occlusal force data are the reference inputs of the simulator. Experimental studies of wear using this simulator demonstrate that integrating the biological feature of combined force/position hybrid control in dental material testing improves the linearity and reduces the variability of results. In addition, it has been shown that present biaxially operated dental wear simulators are likely to provide misleading results in comparative in vitro/in vivo one-contact studies due to neglecting the occlusal sliding motion in one plane which could introduce an error of up to 49% since occlusal sliding motion D and volumetric wear loss V(loss) are proportional.     

Reticulon 4 Is Necessary for Endoplasmic Reticulum Tubulation,STIM1-Orai1 Coupling,and Store-operated Calcium Entry

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca²⁺ levels via SOCE and therefor are less susceptible to Ca²⁺ overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca²⁺ homeostasis.     

Reversal of depressed miduterine arterial flow during hyperthermia-induced respiratory alkalosis

The influence of ambient and arterial PCO2 on miduterine arterial flow of pregnant sheep acutely exposed to hot environments was investigated. Five mixed-breed ewes between 120 and 130 days of gestation were subjected to hot environments (increasing from thermoneutral 23 to 40 degrees C), and arterial blood pH, PCO2, and PO2 were determined at 5-min intervals. Respiratory rate, heart rate, rectal temperature, blood pressure, and miduterine arterial flow were continuously monitored prior to and during elevation of ambient air temperature. When miduterine arterial flow had decreased to 50% of thermoneutral control levels, ambient air CO2 was increased to 2.5%. Elevated ambient inspired CO2 caused a reversal in arterial pH and PCO2 to near thermoneutral levels. Miduterine arterial flow increased to 77% of the control levels following the elevated ambient PCO2 period. Respiratory rate also decreased when ambient CO2 was increased but remained 136% greater than the thermoneutral control level. All other parameters remained near their heat stress (40 degrees C) level during the elevation of ambient CO2. These data indicate that heat-stress-induced depression of miduterine arterial flow is vasoactively regulated, and cause-effect related to both arterial pH and PCO2, and thermoregulatory shunting of blood to heat-dissipating surfaces.     

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.     

Analysis of erythroid differentiation in Friend cells using noninducible variants

A series of Friend cell variants has been isolated by selecting for resistance to different inducers of Friend cell differentiation. This procedure selects for cells which have lost the capacity to differentiate terminally in the presence of inducer. Fluctuation analysis shows that these variants arise during culture and are not induced by the selective conditions. Moreover, mutagenesis of parental cells increases the frequency of occurrence of DMSO-resistant variants. Our evidence suggests that these resistant variants arise by two mechanisms. Some arise spontaneously at a relatively high rate (5 × 10^?5?5 × 10^?6 per cell per generation), but their phenotypes are not necessarily stable on removal of the selective conditions. Stable variants arise spontaneously at a lower frequency which is consistent with a true mutational origin.Screening of these stable resistant variants shows that they have different phenotypes. Some fail to respond to any inducer; others respond to all inducers tested except the one used for selection, whereas others respond to some but not all inducers. Most of the DMSO-resistant variants are noninducible by DMSO for all aspects of Friend cell differentiation tested (that is, globin mRNA, hemoglobin, spectrin and the ability to undergo terminal differentiation). Two variants, however, are inducible for an early marker of differentiation, the erythrocyte membrane protein spectrin, but not for other markers such as hemoglobin, globin RNA or terminal differentiation. This implies that the regulation of the globin pathway can be uncoupled from that of spectrin.     

Two roads diverged: the structure of hydroxymandelate synthase from Amycolatopsis orientalis in complex with 4-hydroxymandelate

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.