Baseline Findings of PreventE4: A Double-Blind Placebo Controlled Clinical Trial Testing High Dose DHA in APOE4 Carriers before the Onset of Dementia.

INTRODUCTION: Lower blood levels of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) are correlated with worse cognitive functions, particularly among APOE ε4 carriers. Whether DHA supplementation in APOE ε4 carriers with limited DHA consumption and dementia risk factors can delay or slow down disease progression when started before the onset of clinical dementia is not known. METHODS: PreventE4 is a double-blind, single site, randomized, placebo-controlled trial in cognitively unimpaired individuals with limited omega-3 consumption and dementia risk factors (n=368). Its objectives are to determine (1) whether carrying the APOE ε4 allele is associated with lower delivery of DHA to the brain; and (2) whether high dose DHA supplementation affects brain imaging biomarkers of AD and cognitive function. RESULTS: 365 cognitively unimpaired individuals between 55 and 80 (mean age 66) were randomized to 2 grams of DHA per day or identically appearing placebo for a period of 2 years. Half the participants were asked to complete lumbar punctures at baseline and 6-month visits to obtain cerebrospinal fluid (CSF). The primary trial outcome measure is the change in CSF DHA to arachidonic acid ratio after 6 months of the intervention (n=181). Secondary trial outcomes include the change in functional and structural connectivity using resting state functional MRI at 24 months (n=365). Exploratory outcomes include the change in Repeatable Battery of the Assessment of Neuropsychological Status at 24 months (n=365). CONCLUSIONS: Findings from PreventE4 will clarify the brain delivery of DHA in individuals carrying the APOE ε4 allele with implications for dementia prevention strategies. Trial was registered as NCT03613844.

Urine dicarboxylic acids change in pre-symptomatic Alzheimer's disease and reflect loss of energy capacity and hippocampal volume.

Non-invasive biomarkers will enable widespread screening and early diagnosis of Alzheimer's disease (AD). We hypothesized that the considerable loss of brain tissue in AD will result in detection of brain lipid components in urine, and that these will change in concert with CSF and brain biomarkers of AD. We examined urine dicarboxylic acids (DCA) of carbon length 3-10 to reflect products of oxidative damage and energy generation or balance that may account for changes in brain function in AD. Mean C4-C5 DCAs were lower and mean C7-C10 DCAs were higher in the urine from AD compared to cognitively healthy (CH) individuals. Moreover, mean C4-C5 DCAs were lower and mean C7-C9 were higher in urine from CH individuals with abnormal compared to normal CSF amyloid and Tau levels; i.e., the apparent urine changes in AD also appeared to be present in CH individuals that have CSF risk factors of early AD pathology. In examining the relationship between urine DCAs and AD biomarkers, we found short chain DCAs positively correlated with CSF Aß42, while C7-C10 DCAs negatively correlated with CSF Aß42 and positively correlated with CSF Tau levels. Furthermore, we found a negative correlation of C7-C10 DCAs with hippocampal volume (p < 0.01), which was not found in the occipital volume. Urine measures of DCAs have an 82% ability to predict cognitively healthy participants with normal CSF amyloid/Tau. These data suggest that urine measures of increased lipoxidation and dysfunctional energy balance reflect early AD pathology from brain and CSF biomarkers. Measures of urine DCAs may contribute to personalized healthcare by indicating AD pathology and may be utilized to explore population wellness or monitor the efficacy of therapies in clinical trials.

Amniotic fluid levels of phospholipase A2 in fetal rats with retinoic acid induced myelomeningocele: the potential "second hit" in neurologic damage.

OBJECTIVES: There is growing evidence of ongoing, in utero neurological damage in fetuses with myelomeningocele (MMC). Phospholipase A2 (PLA2) has known neurotoxic properties and is predominantly present in its secretory isoform (sPLA2) in meconium, the passage of which is increased in MMC fetuses. The objective of this study was to determine if amniotic fluid (AF) levels of PLA2 are elevated in a rat model of MMC. METHODS: Timed pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid (RA) in olive oil at embryonic day 10 (E10). Amniocentesis was performed at multiple gestational time points on MMC fetuses, RA-exposed fetuses without MMC and control fetuses. AF PLA2 levels were analyzed by a fluorescent enzyme activity assay. PLA2 isoforms were determined by measuring activity in the presence of specific inhibitors. RESULTS: There was no difference in AF PLA2 activity between groups on E15. PLA2 activity was significantly increased in MMC fetuses on E17, E19 and E21 (p < 0.001). Secretory PLA2 primarily accounted for the overall greater activity. CONCLUSIONS: PLA2 levels are elevated in the AF of fetal rats with MMC and may contribute to ongoing neural injury. This pathway may be a useful drug target to limit ongoing damage and better preserve neurologic function.

Free amino acid and dipeptide changes in the body fluids from Alzheimer's disease subjects.

Our aim was to determine changes in free amino acid (FAA) and dipeptide (DP) concentrations in probable Alzheimer's disease (pAD) subjects compared with control (CT) subjects using liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2). We recruited gender- and age-matched study participants based on neurological and neuropsychological assessments. We measured FAAs and DPs in cerebrospinal fluid (CSF), plasma and urine using LCMS2 with selected reaction monitoring (SRM). Imidazole-containing FAAs (histidine, methyl-histidine), catecholamines (L-DOPA and dopamine), citrulline, ornithine, glycine and antioxidant DPs (carnosine and anserine) accounted for the major changes between CT and pAD. Carnosine levels were significantly lower in pAD (328.4 +/- 91.31 nmol/dl) than in CT plasma (654.23 +/- 100.61 nmol/dl). In contrast, L-DOPA levels were higher in pAD (1400.84 +/- 253.68) than CT (513.10 +/- 121.61 nmol/dl) plasma. These data underscore the importance of FAA and DP metabolism in the pathogenesis of AD. Since our data show changes in antioxidants, neurotransmitters and their precursors, or FAA associated with urea metabolism in pAD compared with CT, we propose that manipulation of these metabolic pathways may be important in preventing AD progression.

Quantification of free amino acids and dipeptides using isotope dilution liquid chromatography and electrospray ionization tandem mass spectrometry.

Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS2 to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([2H3]-L-Dopa, homoarginine, homophenylalanine, [15N]-Glutamine and [2H3]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05-1 pmol range and is linear over 3-5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 microl CSF, 31 FAAs and 6 DPs in 100 microl plasma, and 23 FAAs and 5 DPs in 200 microl urine. These data demonstrate the potential for using LCMS2 to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.

Structural partitioning, paired-sites models and evolution of the ITS transcript in Syzygium and Myrtaceae.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA are widely used for phylogenetic inference. Several characteristics, including the influence of RNA secondary structure on the mutational dynamics of ITS, may impact on the accuracy of phylogenies estimated from these regions. Here, we develop RNA secondary structure predictions for representatives of the angiosperm family Myrtaceae. On this basis, we assess the utility of structural (stem vs. loop) partitioning, and RNA-specific (paired-sites) models for a 76 taxon Syzygium alignment, and for a broader, family-wide Myrtaceae ITS data set. We use a permutation approach to demonstrate that structural partitioning significantly improves the likelihood of the data. Similarly, models that account for the non-independence of stem-pairs in RNA structure have a higher likelihood than those that do not. The best-fit RNA models for ITS are those that exclude simultaneous double substitutions in stem-pairs, which suggests an absence of strong selection against non-canonical (G.U/U.G) base-pairs at a high proportion of stem-paired sites. We apply the RNA-specific models to the phylogeny of Syzygium and Myrtaceae and contrast these with hypotheses derived using standard 4-state models. There is little practical difference amongst relationships inferred for Syzygium although for Myrtaceae, there are several differences. The RNA-specific approach finds topologies that are less resolved but are more consistent with conventional views of myrtaceous relationships, compared with the 4-state models.

Cross-linked poly(N-acetylethylenimine) as an isoelectric focusing matrix.

Agarose and polyacrylamide are the gels used for most analytical and micropreparative electrophoresis of biopolymers. In an alternative approach that offers different physico-chemical properties from these standard gels, nonionic hydrogels and amphigels composed of poly(N-acetylethylenimine) (PAEI) and a variety of cross-linkers were prepared and used as anticonvective matrices for isoelectric focusing. PAEI was prepared from the ring opening, ionic polymerization of 2-methyl-2-oxazoline. The N-acetyl side chains were hydrolyzed with aqueous sodium hydroxide to produce secondary amine sites which were used for the attachment of cross-linkers. Several cross-linkers were tested for their suitability for electrophoresis, and the cross-linker system based on the Diels-Alder reaction between a furan and maleimide tethered to PAEI gave a moldable gel that can be reversibly converted to a sol at 80 degrees C. This gel was used for isoelectric focusing under both denaturing and nondenaturing conditions. Several protein standards were resolved as well as was achieved with polyacrylamide.

Naturally occurring scrapie in Southdown sheep.

The purpose of this study was to characterize naturally occurring scrapie in the Southdown breed of sheep. Experimental subjects included 4 Southdown ewes admitted to the University of Missouri, College of Veterinary Medicine Large Animal Clinic. All 4 sheep had signs compatible with clinical scrapie. Cerebrospinal fluid (CSF) cell counts ranged from a low of 1 nucleated cell/microL to high of 4 cells/microL with a median of 3 cells/microL. Cerebrospinal protein concentrations ranged from 26 to 78 mg/dL with a median of 53 mg/dL. Immunoassay of the CSF for the 14-3-3 protein yielded positive results in 3 of the 4 sheep. Sequencing of the prion protein (PrP) gene revealed that all 4 sheep were homozygous for glutamine at codon 171 and, hence, were of the QQ genotype. Histopathologic examination of brain stem tissue sections revealed intracytoplasmic neuronal vacuolation and mild spongiform changes in the gray matter neuropil in all 4 ewes. The diagnosis of scrapie was confirmed by immunohistochemical staining for the abnormal PrP Our results suggest that the genetics of scrapie susceptibility are probably similar in Suffolk and Southdown sheep. Positive immunoassay results for the 14-3-3 protein were observed in 3 of the 4 sheep.

Mucosal metabolism in ulcerative colitis and Crohn's disease.

PURPOSE: Colonic mucosal metabolism of butyrate may be impaired in ulcerative colitis. In this study we sought to confirm this observation, to determine if a similar change occurs in Crohn's colitis, and to establish whether a panenteric disorder of butyrate metabolism exists in either condition. METHODS: With use of a microculture technique, mucosal metabolic fluxes of 14[C]-labeled butyrate and 14[C]-labeled glutamine were measured as 14[C] carbon dioxide production in mucosal biopsy specimens from the colon and ileum in patients with ulcerative colitis, Crohn's colitis, and healthy bowel. Results were expressed as pmol/microg biopsy DNA/hour. RESULTS: In the colon the mucosal metabolic fluxes of both butyrate and glutamine are reduced in both ulcerative colitis and Crohn's colitis compared with healthy controls. These changes were most marked in the presence of moderate to severe mucosal inflammation, there being no significant difference in mucosal metabolic flux between mildly inflamed mucosa and healthy controls. In the ileum the mucosal metabolic fluxes of butyrate and glutamine did not differ between healthy controls and those with either ulcerative colitis or Crohn's colitis. CONCLUSIONS: Changes in colonic mucosal metabolism of butyrate and glutamine in inflammatory bowel disease occur as a consequence of the inflammatory process and are not peculiar to ulcerative colitis. Ileal mucosal metabolism is unchanged in ulcerative colitis and Crohn's colitis, indicating the absence of a panenteric abnormality of mucosal metabolism in these two conditions.

Metabolic substrate utilization differs in ileal faecal and urinary reservoirs.

BACKGROUND: Construction of an ileal faecal or urinary reservoir profoundly alters ileal luminal ecology and availability of mucosal metabolic substrates. The aims of this study were to measure mucosal metabolic flux of butyrate and glutamine in histologically normal (control) ileum and to determine the effect of reservoir construction on metabolic fluxes in patients with ileal pouch-anal anastomosis and ileocystoplasty. METHODS: Endoscopic biopsy samples were obtained from normal ileum (n = 10), ileum of patients with ulcerative colitis (n = 10), ileal pouch-anal anastomosis (n = 7), ileocystoplasty (n = 7) and ileal conduit (n = 7). Using a closed microculture technique, biopsy utilization of 14C-labelled butyrate and glutamine was measured as [14C]carbon dioxide production. Biopsy DNA content was measured and [14C]carbon dioxide evolution expressed as picomoles [14C]carbon dioxide per microgram DNA per hour. RESULTS: The metabolic flux of both butyrate and glutamine was reduced in ileal pouch mucosa compared with that of ileal mucosa in patients with ulcerative colitis. In contrast, the metabolic flux of buyrate alone was reduced in ileal mucosa from ileocystoplasty and ileal conduit compared with that in normal ileal mucosa, while the metabolic flux of glutamine remained unchanged. CONCLUSION: Ileal mucosal metabolic fluxes measured in vitro are altered by changing luminal ecology in vivo. These changes may affect the health and mucosal integrity of ileum used to construct these reservoirs.

Covalent variation is a general property of transcription factors in the sea urchin embryo.

Nine different transcription factors of the Strongylocentrotus purpuratus embryo, belonging to diverse structural families, were examined by high-resolution, two-dimensional gel electrophoresis. These factors had all been cloned previously, and antibodies against the recombinant proteins were available. The factors were visualized immunologically in nuclear extracts from 24-hour blastula-stage embryos. Remarkably, every one of the nine factors displayed multiple charge variants. Three factors, SpZ12-1, SpP3A2, and SpRunt-1, were studied at different stages of embryonic development. The prevalence and distribution of the variant isoforms of all three factors differed at each stage examined; and in all cases the complexity of the variants was greatest in the 24-hour blastula-stage extracts. The most complex set of variants was observed for SpP3A2, and phosphatase treatment demonstrated that some but not all, of the covalent modifications defining these variants are phosphorylations. As the transcription factors were chosen for this study merely on the basis of the availability of antibodies, we conclude that deployment of transcription factors in sea urchin embryos generally involves their covalent modification.

The assay development of a molecular marker for transmissible spongiform encephalopathies.

The transmissible spongiform encephalopathies are a group of neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Two-dimensional electrophoresis of proteins was previously used to identify two marker proteins, 130/131, which are selectively present in the cerebrospinal fluid (CSF) of patients with CJD and not in patients with other dementias. The recent characterization of these proteins by amino acid sequencing has identified them as members of the 14-3-3 family of proteins. Polyclonal antibodies against 14-3-3 (all isoforms), 14-3-3 gamma, 14-3-3 beta, and 14-3-3 theta are immunoreactive with a 30 kDa marker band from CJD CSF. Silver staining of two-dimensional electrophoresis separated BSE CSF proteins does not identify a similar marker. However, 14-3-3 immunoreactivity is found in cattle CSF when these proteins are blotted to polyvinylidene difluoride but not when blotted to nitrocellulose.

SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos.

The CyIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme.

The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies.

BACKGROUND: There is no practical and reliable premortem test for Creutzfeldt-Jakob disease and the related transmissible spongiform encephalopathies. Two proteins, designated 130 and 131, which have been detected in low concentrations in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease, appear to be sensitive and specific markers for the disease. Attempts to identify these proteins, however, have been unsuccessful. We hypothesized that they may be present in the normal brain. METHODS: We detected proteins 130 and 131 in normal human brain, partially sequenced their amino acids, and found that they matched the brain protein known as 14-3-3. We then developed a simple, rapid immunoassay for this protein and tested it in cerebrospinal fluid samples from 71 humans and 30 animals with spongiform encephalopathies and in control samples from 186 humans and 94 animals. RESULTS: The immunoassay detected the 14-3-3 protein in cerebrospinal fluid from 68 of the 71 patients with Creutzfeldt-Jakob disease (96 percent, 95 percent confidence interval, 92 to 99 percent). Among 94 patients with other dementias, the specificity was 96 percent. If one excludes the three patients with dementia who had strokes within one month before testing, the specificity was 99 percent. The test was positive in 12 of 24 patients with viral encephalitis. In animals the sensitivity of the assay was 87 percent and the specificity was 99 percent. CONCLUSION: In patients with dementia, a positive immunoassay for the 14-3-3 brain protein in cerebrospinal fluid strongly supports a diagnosis of Creutzfeldt-Jakob disease. This finding, however, does not support the use of the test in patients without clinically evident dementia.

Two-dimensional electrophoresis of proteins as a tool in the metabolic engineering of cell cycle regulation.

Metabolic engineering of cell cycle regulation addresses important biotechnological problems about serum removal from animal cell culture systems. Chinese hamster ovary cells stimulated to grow by fetal calf serum, insulin, or basic fibroblast growth factor were studied by two-dimensional electrophoresis (2DE) and the resulting protein expression patterns were analyzed. Detailed 2DE protocols are provided and at least 24 gene products are identified which may play an important role in growth factor signaling. Moreover, a correlation between the expression of three proteins (cyclin D1, cyclin E, and E2F-1) and mitogenic strength was found.

SpRunt-1, a new member of the runt domain family of transcription factors, is a positive regulator of the aboral ectoderm-specific CyIIIA gene in sea urchin embryos.

In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with a cis-regulatory target site of the CyIIIa gene. Here we show that this target site, the P7I site, is required for normal embryonic activation of CyIIIa x CAT reporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a "runt domain" that is closely homologous to those of Drosophila and mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation of CyIIIa that occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.