The mTOR inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells.

In Ph-positive (Ph(+)) leukemia, the quiescent cell state is one of the reasons for resistance to the BCR-ABL-kinase inhibitor, imatinib. In order to examine the mechanisms of resistance due to quiescence and the effect of the mammalian target of rapamycin inhibitor, everolimus, for such a resistant population, we used Ph(+) acute lymphoblastic leukemia patient cells serially xenotransplanted into NOD/SCID/IL2rγ(null) (NOG) mice. Spleen cells from leukemic mice showed a higher percentage of slow-cycling G(0) cells in the CD34(+)CD38(-) population compared with the CD34(+)CD38(+) and CD34(-) populations. After ex vivo imatinib treatment, more residual cells were observed in the CD34(+)CD38(-) population than in the other populations. Although slow-cycling G(0) cells were insensitive to imatinib in spite of BCR-ABL and CrkL dephosphorylation, combination treatment with everolimus induced substantial cell death, including that of the CD34(+)CD38(-) population, with p70-S6 K dephosphorylation and decrease of MCL-1 expression. The leukemic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse system with the in vivo combination treatment with imatinib and everolimus showed a decrease of tumor burden including CD34(+) cells. These results imply that treatment with everolimus can overcome resistance to imatinib in Ph(+) leukemia due to quiescence.

Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells.

The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. We show that expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. We show that Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. Our experiments reveal that cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient.

Epithelial-mesenchymal signaling during the regionalization of the chick gut.

The development of the vertebrate gut requires signaling between the endoderm and mesoderm for establishing its normal anteroposterior (AP) axis and for tissue-specific differentiation. Factors implicated in positional specification of the AP regions of the gut include endodermally expressed Sonic hedgehog (Shh), mesodermally expressed Bmp4 and members of the Hox gene family. We have investigated the roles of these factors during AP regional specification of the chick embryonic gut. Early in gut development, the endoderm sends inductive signals to the mesoderm. Shh has been implicated as one of these signals. We find a differential response to exposure of the inductive influence of Shh along the AP axis of the gut. Virally mediated misexpression of Shh results in ectopic upregulation of its receptor Ptc and a cellular proliferation throughout the gut mesoderm. Although ectopic Shh can induce Bmp4 in the mesoderm of the midgut and hindgut, Bmp4 is not induced in the stomach region of the foregut. The stomach region has a thicker layer of mesoderm than the rest of the gut suggesting that the normal function of Bmp4 could be to limit mesodermal growth in the non-stomach regions of the gut. Ectopic Bmp4 expression in the stomach results in a reduction of the mesodermal component consistent with this hypothesis. In addition to the regional restriction on Bmp4 induction, Shh can only induce Hoxd-13 in the mesoderm of the hindgut. These findings suggest that a prepattern exists in the primitive gut mesoderm prior to expression of Shh in the endoderm. The gut mesoderm is subsequently responsible for inducing region-specific differentiation of its overlying endoderm. We tested the role of Hoxd-13, normally restricted in its mesodermal expression to the most posterior region of the hindgut (cloaca), in controlling adjacent endodermal differentiation. When virally mediated Hoxd-13 is misexpressed in the primitive midgut mesoderm, there is a transformation of the endoderm to the morphology and mucin content of the hindgut. Thus, the positionally restricted expression of a Hox gene in the gut mesoderm influences the inductive signaling that leads to regionally specific differentiation of gut endoderm.

Reorganization of the spinal dorsal horn in models of chronic pain: correlation with behaviour.

Central reorganization is known to occur in chronic pain models resulting from peripheral injury. Systematic analysis of anatomical and behavioural changes and a comparison of these changes between different models over an extended time course has not been reported. We address this issue by quantifying alterations in markers known to be associated with central reorganization in three models of peripheral injury: complete Freund's adjuvant induced inflammation of the hindpaw, chronic constriction of the sciatic nerve, and tight ligation of the sciatic nerve. Hyperalgesic behaviour to thermal and mechanical stimuli was quantified at four, seven, 14, 28 days post-injury. Distribution and immunodensity changes of the mu-opioid receptor, the neurokinin-1 receptor, and brain nitric oxide synthase distribution were assessed in the superficial dorsal horn, laminae I-II, of the lumbar spinal cord of the rat. Reorganization and behavioural changes were quantified as a per cent change (ipsilateral versus contralateral) and examined together over the duration of the experiment. Chronic constriction injury and inflammation both produced hyperalgesic behaviour in the hindpaw ipsilateral to injury. Decreases in thermal and mechanical withdrawal latencies were maximal at day 4. Complete Freund's adjuvant-treated animals displayed a 25.5%+/-3.8% decline in thermal withdrawal latency and 84.1%+/-8.0% decline in mechanical withdrawal latency. Chronic constriction of the sciatic nerve resulted in an decrease in thermal and mechanical withdrawal latencies, 27.9%+/-3.3%, 90.5%+/-4.4%, respectively. Tight ligation of the sciatic nerve resulted in early increases in the latency of withdrawal that were maximal at seven days 40.7%+/-8.4% for thermal stimulus and at four days 417%+/-5.8% for mechanical stimulus, consistent with deafferentation. The greatest changes in immunolabelling were always found at L4-L5 spinal level, corresponding to the entry zone of sciatic afferents. Mu-opioid receptor immunodensities increased in the dorsal horn ipsilateral to the treated side up to a maximum of 38.3%+/-5.6% at day 7 with inflammation and up to 26.3%+/-3.2%, at day 14 with chronic constriction injury. Mu-opioid receptor immunodensities decreased maximally by 20.0%+/-2.1% at day 4 in the tight ligature model. Significant differences in mu-opioid receptor immunolabelling persisted at day 28 for neuropathic models, at which time there was an absence of significant hyperalgesic behaviour in any group. The number of brain nitric oxide synthase-positive cells decreased at seven days by a maximum of 45.3%+/-5.1% and 59.0%+/-5.2%, respectively, in animals with chronic constriction injury or tight ligature. This decline in immunolabelled brain nitric oxide synthase cells in the dorsal horn ipsilateral to injury persisted at day 28. No significant alteration in brain nitric oxide synthase immunolabelling was found in association with inflammation of the hindpaw. Inducible nitric oxide synthase was not detected in the dorsal horn at any time during the experiment in either tissue of treated or control rats. Neurokinin-1 receptor immunodensity consistently increased ipsilateral to injury irrespective of the type of injury, and, of the three markers, paralleled behaviour most closely. Changes were maximal for inflammation at four days (75.2%+/-9.3%), for chronic constriction injury at four days (85.1%+/-14.6%) and for tight ligature at 14 days (85.7%+/-11.4%). Comparison of behavioural and anatomical data demonstrates that the peak hyperalgesia is concomitant with the greatest increase in neurokinin-1 receptor immunodensity ipsilateral to the injury. The increase in mu-opioid receptor immunodensity parallels behaviour but with a delayed time course, peaking as hyperalgesia abates, except in the case of tight ligature animals where the decrease in immunolabelling appears permanent. (ABSTRACT TRUNCATED)

Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth.

Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. Here we report the misexpression phenotype for Hoxd-13 and compare it to the misexpression phenotype of Hoxd-11. Hoxd-13 misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly (Muragaki, Y., Mundlos, S., Upton, J. and Olsen, B. R. (1996) Science 272, 548-551). N-terminal truncations of Hoxd-13 which lack this repeat were constructed and were found to produce a similar, although slightly milder, misexpression phenotype than the full-length Hoxd-13. The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 misexpression are late phenotypes that first become apparent during the growth phase of the bones. Analysis of tritiated thymidine uptake by the tibia and fibula demonstrates that Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. We propose a model in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes.

The inhibition of human salivary alpha-amylase by type II alpha-amylase inhibitor from Triticum aestivum is competitive, slow and tight-binding.

A kinetic analysis of the inhibition of human salivary alpha-amylase (EC 3.2.1.1) by wheat seed (Triticum aestivum) type II alpha-amylase inhibitor revealed the inhibition was slow and tight-binding. The inhibition was competitive with an inhibition binding constant of the alpha-amylase inhibitor for alpha-amylase of 0.29 nM. The KM of alpha-amylase for soluble starch (calculated per mole of alpha-1,4 linked maltose residues) was 5.87 mM.

Identification of polymorphic simple sequence repeats in the genome of the zebrafish.

The zebrafish has drawn a great deal of attention as a developmental system because it offers the ability to combine excellent embryology and genetics. Here, we report that simple sequence repeats are abundant in the zebrafish genome and are highly polymorphic between two outbred lines, making them useful markers for the construction of a genetic map of this organism.