Role of common and rare APP DNA sequence variants in Alzheimer disease.

OBJECTIVES: More than 30 different rare mutations, including copy number variants (CNVs), in the amyloid precursor protein gene (APP) cause early-onset familial Alzheimer disease (EOFAD), whereas the contribution of common APP variants to disease risk remains controversial. In this study we systematically assessed the role of both rare and common APP DNA variants in Alzheimer disease (AD) families. METHODS: Families with EOFAD genetically linked to the APP region were screened for missense mutations and locus duplications of APP. Further, using genome-wide DNA microarray data, we examined the APP locus for CNVs in a total of 797 additional early- and late-onset AD pedigrees. Finally, 423 single nucleotide polymorphisms (SNPs) in the APP locus, including 2 promoter polymorphisms previously associated with AD risk, were tested in up to 4,200 individuals from multiplex AD families. RESULTS: Analyses of 8 21q21-linked families revealed one family carrying a nonsynonymous mutation in exon 17 (Val717Leu) and another family with a partially penetrant 3.5-Mb locus duplication encompassing APP. CNV analysis in the APP locus revealed an additional family carrying a fully penetrant 380-kb duplication, merely spanning APP. Last, contrary to previous reports, association analyses of more than 400 different SNPs in or near APP failed to show significant effects on AD risk. CONCLUSION: Our study shows that APP mutations and locus duplications are a very rare cause of EOFAD and that the contribution of common APP variants to AD susceptibility is insignificant. Furthermore, duplications of APP may not be fully penetrant, possibly indicating the existence of hitherto unknown protective genetic factors.

Spontaneously hypertensive, Wistar Kyoto and Sprague-Dawley rats differ in their use of place and response strategies in the water radial arm maze.

This study further characterises the use of mnemonic systems in the spontaneously hypertensive rat (SHR), which is frequently used as a rodent model of attention deficit hyperactivity disorder. The objective of this study was to assess the preference of male SHR, Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats for a place or response strategy when trained on an ambiguous T-maze task, and also to examine whether all strains acquired information about both strategies during ambiguous training, regardless of their preferred strategy. In the first experiment, SHR and WKY showed a preference for a response strategy on the ambiguous T-maze task; in contrast, SD displayed a preference for a place strategy. In the second experiment, all strains demonstrated that they learned information about both the response and place strategies during ambiguous training. However, on a conditioned place preference test SHR did not display as strong a preference for the place arm as WKY and SD. This finding supports previous research in a conditioned cue preference test, in which SHR did not display a preference for the cue associated with the platform. These observations that the strains differ with respect to behavioural strategy in a learning task suggest that they differ in the underlying neural circuitry that serves goal-directed behaviour, and are consistent with SHR having deficits associated with the nucleus accumbens.

Interpreting the effects of small uncharged solutes on protein-folding equilibria.

Proteins are designed to function in environments crowded by cosolutes, but most studies of protein equilibria are conducted in dilute solution. While there is no doubt that crowding changes protein equilibria, interpretations of the changes remain controversial. This review combines experimental observations on the effect of small uncharged cosolutes (mostly sugars) on protein stability with a discussion of the thermodynamics of cosolute-induced nonideality and critical assessments of the most commonly applied interpretations. Despite the controversy surrounding the most appropriate manner for interpreting these effects of thermodynamic nonideality arising from the presence of small cosolutes, experimental advantage may still be taken of the ability of the cosolute effect to promote not only protein stabilization but also protein self-association and complex formation between dissimilar reactants. This phenomenon clearly has potential ramifications in the cell, where the crowded environment could well induce the same effects.

Evidence for genetic linkage of Alzheimer's disease to chromosome 10q.

Recent studies suggest that insulin-degrading enzyme (IDE) in neurons and microglia degrades Abeta, the principal component of beta-amyloid and one of the neuropathological hallmarks of Alzheimer's disease (AD). We performed parametric and nonparametric linkage analyses of seven genetic markers on chromosome 10q, six of which map near the IDE gene, in 435 multiplex AD families. These analyses revealed significant evidence of linkage for adjacent markers (D10S1671, D10S583, D10S1710, and D10S566), which was most pronounced in late-onset families. Furthermore, we found evidence for allele-specific association between the putative disease locus and marker D10S583, which has recently been located within 195 kilobases of the IDE gene.

Presenilin-mediated modulation of capacitative calcium entry.

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction.

The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the alpha-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent alpha-crystallin cross-linking. 3HK- or 3HAA-modifed alpha-crystallin showed enhanced redox activity in comparison to unmodified alpha-crystallin or ascorbate-modified alpha-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as alpha-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.

Osmolyte-induced changes in protein conformational equilibria.

Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria.

Renovascular disease.

The gold standard for the diagnosis of renal artery stenosis is angiography, with response to treatment the proof of its significance. Non-invasive methods of investigation are required and are now available including functional imaging, ultrasound, CT and MR angiography and the merits and limitations of these tests are discussed.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

Cystic kidney disease presenting in infancy.

AIM: The clinical, histological and imaging findings of 12 children with ultrasound features of severe renal cystic disease presenting in the first year of life were reviewed. METHODS AND RESULTS: Two children had cystic dysplasia and four had autosomal dominant polycystic disease. Two had a malformation syndrome, one a variant of Meckel syndrome and the other Bardet Biedl syndrome. One had autosomal recessive polycystic disease and in three there was no final diagnosis. Intravenous urography gave non-specific information. In six cases clinical findings combined with imaging established a diagnosis. Diagnosis was established by biopsy in two and gave supportive evidence in one. Outlook for renal function is variable. One child has had a transplant and one is on dialysis awaiting a transplant. Three have a degree of renal failure and one has died. Six have normal renal function. Renal cystic disease is the common pathway for a heterogeneous group of disorders as shown in these children. CONCLUSION: It is emphasized that a specific diagnosis could not be made from the renal sonographic appearances alone, nor could any prognostic implications for renal function be made. Contrast retention on intravenous urography was also insufficiently specific to be of value. Ultrasound of the parents was the most useful imaging procedure and should be done in all cases.

Partially formed native tertiary interactions in the A-state of cytochrome c.

Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state.

Sugar-induced molten-globule model.

Proteins denature at low pH because of intramolecular electrostatic repulsions. The addition of salt partially overcomes this repulsion for some proteins, yielding a collapsed conformation called the A-state. A-states have characteristics expected for the molten globule, a notional kinetic protein folding intermediate. Here we show that the addition of neutral sugars to solutions of acid-denatured equine ferricytochrome c induces formation of the A-state in the absence of added salt. We characterized the structure and stability of the sugar-induced A-state with circular dichroism spectropolarimetry (CD) and NMR-monitored hydrogen-deuterium exchange experiments. We also examined the stability of the sugar-induced A-state as a function of sugar size and concentration. The results are interpreted using several models and we conclude that the stabilizing effect is consistent with increased steric repulsion between the protein and the sugar solutions.

Ultrasound examination of neonate's hip joints.

In a mass screening project, 872 neonates were examined clinically and by ultrasound. Hip joint stability was assessed in the ultrasound stress test showing a normal or physiologic instability, in the range of 0 to 2 mm. Along with the dynamic examination, a modified Graf's method was used. All obtained sonograms were submitted to careful assessment by one of authors, including regular measurement of alpha and beta angles. This relatively small group in the low-risk population served as a model of normality. White newborns compared with an black Caribbean group did not show the presence of apparent primary acetabular dysplasia. Both methods--dynamic ultrasound test and Graf's scanning--seemed to us to be valuable.

The incidence of transient renal medullary hyperechogenicity in neonatal ultrasound examination.

A prospective ultrasound study of the urinary tracts of 85 neonates (64 term, 21 preterm) was performed to assess the incidence of transient renal medullary hyperechogenicity (RMH) in the first week of life. None of the neonates examined had evidence of renal dysfunction. Echogenic material was observed in the renal papillae/calyces, ureter, or bladder of 33 of the 64 term babies, but in the bladder of only one pre-term infant. The distribution of the echogenicity differs from that seen in medullary hyperechogenicity due to crystal deposition, suggesting that calyceal involvement is a common feature. Follow-up ultrasound scans at 10-14 days were possible in eight of the term neonates and demonstrated complete resolution of the RMH. The aetiology of transient neonatal RMH is unclear, although it may be related to protein cast deposition in the renal tubules. RMH may rarely be associated with transient renal dysfunction, but in healthy neonates should be recognized as a normal variant.

The ultrasound appearances of neonatal renal vein thrombosis.

Renal vein thrombosis (RVT) is the most frequently occurring vascular condition in the new-born kidney. The predisposing factors include dehydration, sepsis, birth asphyxia, maternal diabetes, polycythaemia and the presence of an indwelling umbilical venous catheter. (RVT) may present clinically with a flank mass, haematuria, hypertension or renal failure. Many imaging modalities have been employed, but ultrasound is the technique most commonly used in the evaluation of neonates with suspected RVT. Thrombosis commences in the small renal veins and subsequently propagates via larger interlobar veins to the main renal vein and inferior vena cava (IVC). The ultrasound appearances depend upon the stage at which the examination is performed and extent of the thrombus. Initially, the interlobular and interlobar thrombus appears as highly echogenic streaks. These streaks commence in a peripheral, focal segment of the involved kidney and only persist for a few days. In the first week the affected kidney swells and becomes echogenic with prominent echopoor medullary pyramids. Later, the swelling increases and the kidney becomes heterogenous with loss of corticomedullary differentiation. Grey scale ultrasound readily demonstrates thrombus within the renal vein and IVC. Adrenal haemorrhage is a recognized association and may be identified ultrasonically. Colour Doppler scanning provides additional information. In the early stages of RVT, colour Doppler may demonstrate absent intrarenal and renal venous flow. Ultimately, the kidney may recover, show focal scarring or become atrophic. Thus, ultrasound provides an accessible and reliable tool in the assessment of suspected neonatal RVT.

Unusual effects of an engineered disulfide on global and local protein stability.

The global and local stabilities of a eukaryotic ferricytochrome c variant with an engineered disulfide are examined. The disulfide connects position 20, which is usually a valine, to position 102, which is usually a threonine. The cross-linked variant is approximately 1.2 kcal mol-1 less stable than the wild-type protein at 298 K, pH 4.6, in H2O and D2O. Circular dichroism studies show that the decreased stability results from structure-induced stabilization of the denatured state [Betz, S. F., & Pielak, G. J. (1992) Biochemistry 31, 12337-12344]. Here, we use proton chemical shift, paramagnetic shift, and amide proton exchange data to obtain atomic level structural and energetic information. Chemical and paramagnetic shift data indicate only minor native state structural changes. Local stability is obtained from amide proton-deuterium exchange data, using model peptide intrinsic exchange rates. As expected, the exchange data indicate that cross-link incorporation decreases the majority of local stabilities. Near the cross-link, however, local stability seems to increase despite the overall global stability decrease. Furthermore, local stability changes for hydrophobic core residues seem to be greater than the global stability change. We interpret these observations as cross-link-induced changes in exchange competent states and relate them to changes in the denatured state.

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

Effects of recombinant human growth hormone on renal function in children with renal transplants.

OBJECTIVE: To provide accurate measurement of renal function during treatment with recombinant human growth hormone (rhGH). METHODS: We measured glomerular filtration rate and effective renal plasma flow by clearance of inulin and para-aminohippuric acid before rhGH therapy, after 1 week, and then at 6-month intervals for up to 2 years of treatment in 16 children (mean (SD) age = 13.1 (2.2) years; glomerular filtration rate = 52 (27) ml/min per 1.73 m2). The mean (SD) time from transplantation was 6.5 (3.6) years. RESULTS: Linear growth velocity during rhGH therapy increased from 4.0 (1.8) to 8.8 (2.6) cm/yr (p < 0.0001). One child was withdrawn after 9 months because of abnormal glucose tolerance, and another child received a second renal transplant after 18 months. Glomerular filtration rate increased to 57 (29) ml/min per 1.73 m2 at 1 week (p = 0.004), remained improved at 6 months (63 (30); p = 0.013), but was not significantly better at 1 year (59 (33)). Effective renal plasma flow on day 1 was 237 (127) ml/min per 1.73 m2 and was unchanged on day 8 (244 (123)), at 6 months (271 (149)), and after 1 year (269 (157)). During the study there was no significant change in filtration fraction, blood pressure, or kidney volume, and excretion of microalbumin and N-acetylglucosaminidase was unaltered. There was one rejection episode per 14.8 patient-months in the year before treatment, 1 per 18.9 patient-months during the first year of treatment, and 1 per 13 patient-months during the second year of rhGH therapy. CONCLUSION: Treatment with rhGH improves growth in children with renal transplants. Glomerular filtration rate was increased after 1 week and 6 months of rhGH therapy but returned to baseline values thereafter. The data indicate the need for long-term follow-up of children with renal transplants who are receiving rhGH.