Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains

We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q. In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub- domains with entirely different patterns of homology to other chromosome ends. The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions. The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends. A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends. Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non- homologous, chromosomes. These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.      

Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase

The neurochemical identity of ascending putative cholinergic pathways from the rat basal forebrain was investigated employing a method for simultaneously visualizing choline acetyltransferase immunoreactivity and retrogradely transported horseradish peroxidase-conjugated wheatgerm agglutinin. This histochemical procedure revealed three distinct populations of neurons: (1) cells which stained only for choline acetyltransferase immunoreactivity; (2) cells which stained only for retrograde tracer and (3) cells which stained simultaneously for choline acetyltransferase immunoreactivity and retrograde tracer. The results demonstrated that this projection is topographically organized and consists of both cholinergic and noncholinergic components. The relative contribution of each component varied with the telencephalic target area as follows: the olfactory bulb receives a projection from cells of the horizontal limb nucleus, 10-20% of which are cholinergic (Ch3); the hippocampal formation receives afferents from cells of the medial septal and vertical limb nuclei, 35-45% of which are cholinergic (Ch1 and Ch2); and the cortical mantle receives afferents primarily from cells within the substantia innominata-nucleus basalis complex, 80-90% of which are cholinergic (Ch4). The topographical organization of Ch4 projections is not as completely differentiated as we have previously observed in the primate.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Phorbol ester induced 1,2-diacylglycerol accumulation and protein phosphorylation in C3H/10T1/2 mouse embryo cells

Within 10 min of addition of the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate(TPA) to C3H/10T1/2 mouse embryo fibroblasts, there is a two-foldincrease in the level of cellular 1,2-diacylglycerol levelscompared to controls. This increase in 1,2-diacylglycerol isdependent on the concentration of TPA added to the cell culturemedium. The ability of macrocyclic diterpenes to induce 1,2-diacylglycerolaccumulation correlated with their tumor promoting activityexcept for mezerein. The accumulation of 1,2-diacylglycerolin response to TPA was not blocked by a concentration of cycloheximidesufficient to inhibit protein synthesis by 95%. These data supportour previous suggestion that TPA activates a phospholipase C.During the same time period, TPA increased protein phosphorylationin both quiescent and growing cells. Proteins of mol. wt. 50000, 45 000, 35 000 and 27 000 are markedly phosphorylated inresponse to TPA in both growing and quiescent cultures. Therelationship of these phosphorylated proteins to a Ca²⁺ phospholipidactivated protein kinase remains to be determined.     

Multiple cerebral metastases: detectability with Gd-DTPA-enhanced MR imaging

Sixteen patients with suspected cerebral metastases were studied with magnetic resonance (MR) imaging before and after the intravenous administration of 0.1 mmol/kg of gadolinium diethylenetriaminepenta-acetic acid. The images were interpreted blindly by two neuroradiologists; all clinical, radiologic (computed tomographic and MR imaging), and pathologic data were reviewed to arrive at a final "best diagnosis," which was then compared with the prior blinded interpretations. Of seven patients found to have multiple metastases, six (86%) had at least one tumor nodule depicted by postinfusion MR imaging that was missed by one or both observers on review of preinfusion images alone. Lesions missed on preinfusion studies were usually small nodules hidden by or not detected next to regions of high-signal edema thought to be related to the adjacent tumor nodule. The authors believe that contrast enhancement improves detection of metastatic foci with MR imaging and that the findings indicate broader implications for the detection of multiple lesions from other causes.     

Cholinesterases colocalize with sites of neurofibrillary degeneration in aged and Alzheimer's brains

Acetylcholinesterase and butyrylcholinesterase have been associated with structures undergoing neurofibrillary degeneration, as well as with all types of senile plaques, in non-demented aged and Alzheimer's brains. At the electron microscope level, the reaction product of both enzymes, appeared to decorate paired helical filaments, straight filaments and A4 amyloid fibrils. Recent studies showed that cholinesterases were associated with amyloid at early stages, e.g., in diffuse plaques. In the present study, the interrelationship of cholinesterases to structures undergoing neurofibrillary degeneration was analyzed further. Tau immunoreactivity was compared to the staining pattern observed with the two esterases. Double protocols consecutively performed on the same sections, and counterstaining with thioflavin-S, confirmed the presence of cholinesterases in all structures with neurofibrillary degeneration. The conclusion that cholinesterases consistently colocalize with both neurofibrillary bundles and A4 amyloid fibrils at all stages of their accumulation, allows us to speculate on the possible role that these enzymes may play in either the formation or the consolidation of fibrillary aggregates.Supported by Fondo de Investigaciones Sanitarias de la Seguridad Social No. 93/0198, Spain     

Phorbol esters stimulate the rapid release of choline from prelabelled cells

Evidence implicating the cell surface membrane as the primarysite of action of phorbol ester tumor promoters has led us tocharacterize early changes in phospholipid metabolism whichoccur in response to these compounds. When C3H10T mouse embryofibroblasts were incubated with [³H]choline for 24 h, {smalltilde}78% of the cell associated material was found in phosphatidylcholine and the remainder in sphingomyelin and the acid solublepool. Within 5 min of exposure of these prelabelled cells tothe potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(PPD) the release of water soluble [³H] metabolites from cellsinto the medium was enhanced 2-fold and by 60–120 minthe release was 2–5 times that found in control cultures.The released material was identified by chromatography as cholineand phosphoryl choline. Evidence was obtained that this materialwas not derived exclusively from the acid soluble pool or fromthe degradation of labelled sphingomyelin. Choline metaboliterelease was concentration dependent between 10^–8 and 10^–7M TPA and was not associated with cell toxicity. Phorbol-12,13-didecanoate(PDD) was also active but 4PDD, which is not a tumor promoter,was inactive. Neither cyclohexlmide (4–40 µg/ml)nor cordycepln (4–40 µg/ml) blocked the TPA inducedrelease. The release was, however, temperature sensitive anddid not occur at 4°C. TPA did not induce the release of[³H]inositol from prelabelled phosphatidyl inositol. Although,the calcium ionophore A23187 [GenBank] induced the release of arachidonicacid from prelabelled cells it did not induce choline release.In addition, 5,8,11,14-eicosatetraynoic acid, which inhibitsboth lipoxygenase and cyclooxygenase activity, did not inhibitTPA stimulation of choline release. We propose that the bindingof TPA to cell surface receptors leads to a rapid activationof membrane associated phospholipase(s) that specifically degradephosphatidyl choline. This effect may be analogous to the abilityof other agonists to activate degradation of phosphatidyl inositol.