The role of test accuracy in predicting acceptance of genetic susceptibility testing for Alzheimer's disease

A survey questionnaire regarding perceptions of risk and genetic susceptibility to Alzheimer's disease (AD) was completed by 518 offspring of AD cases from families with multiple affected, ascertained as part of a genetic linkage study of late onset AD. The questionnaire focused on respondents' perceptions of their own risk for AD as well as on the properties of real and hypothetical susceptibility tests, including error rates for false-positive and false-negative test results. Our findings showed that about 20% of the sample would refuse a susceptibility test with zero error rates, about 40% would accept tests with very high error rates in both directions, and the remainder would exercise some discrimination. Acceptance of high test error rates was significantly associated with male gender, low education, and high perceived lifetime risk of AD. In a previous paper related to this work, we showed that physicians caring for these families exercised much more discrimination in judging the acceptability of genetic tests they would offer to these same respondents. The findings show that there is a pressing need to educate the public, particularly those with relatives affected by a complex disease, to expect standards of accuracy for genetic tests comparable to those that prevail in other diagnostic and prognostic testing efforts in the broad field of clinical medicine.     

Histone deacetylase activity is required for embryonic stem cell differentiation

Mammalian development requires commitment of cells to restricted lineages, which requires epigenetic regulation of chromatin structure. Epigenetic modifications were examined during in vitro differentiation of murine embryonic stem (ES) cells. Global histone acetylation, a euchromatin marker, declines dramatically within 1 day of differentiation induction and partially rebounds by day 2. Histone H3-Lys9 methylation, a heterochromatin marker, increases during in vitro differentiation. Conversely, the euchromatin marker H3-Lys4 methylation transiently decreases, then increases to undifferentiated levels by day 4, and decreases by day 6. Global cytosine methylation, another heterochromatin marker, increases slightly during ES cell differentiation. Chromatin structure of the Oct4 and Brachyury gene promoters is modulated in concert with their pattern of expression during ES cell differentiation. Importantly, prevention of global histone deacetylation by treatment with trichostatin A prevents ES cell differentiation. Hence, ES cells undergo functionally important global and gene-specific remodeling of chromatin structure during in vitro differentiation. genesis 38:32-38, 2004.     

Structural analysis of DNA-PKcs: modelling of the repeat units and insights into the detailed molecular architecture

DNA-dependent protein kinase (DNA-PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA-PK (DNA-PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of approximately 4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA-PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA-PKcs protein and identifies regions of interest for further experimental studies.     

Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin

ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.     

Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

Catecholamines act via a beta-adrenergic receptor to maintain fetal heart rate and survival

Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereo-specific manner when administered in the dam's drinking water. An alpha-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of alpha-ARs with 10 microM phentolamine or beta-ARs with 10 microM bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, beta-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the alpha-AR antagonist phentolamine had no effect. These data suggest that beta-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through beta-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia.     

Probing the activation site of ribonuclease L with new N6-substituted 2',5'-adenylate trimers

2-5A trimer [5'-monophosphoryladenylyl(2'-5')adenylyl(2'-5')adenosine] activates RNase L. While the 5'-terminal and 2'-terminal adenosine N(6)-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5'-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5'-O-dimethoxytrityl-3'-O-(tert-butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine 2'-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N(6)-substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5'A2'p5'A2'p5'A. Thus, the spatial domain about this N(6)-amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.     

A peptide from heat shock protein 60 is the dominant peptide bound to Qa-1 in the absence of the MHC class Ia leader sequence peptide Qdm

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.     

Isofurans,but not F2-isoprostanes,are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease

F2-isoprostanes (F2-IsoPs) are well-established sensitive and specific markers of oxidative stress in vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2-IsoPs, their formation is favored when oxygen tension is increased in vitro or in vivo. Mitochondrial dysfunction in Parkinson's disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2-IsoPs and IsoFs. In this study, we attempted to compare the levels of F2-IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age-matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). The results demonstrated that IsoFs but not F2-IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2-IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age-matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.