Novel Alzheimer Disease Risk Loci and Pathways in African American Individuals Using the African Genome Resources Panel: A Meta-analysis.

Importance: Compared with non-Hispanic White individuals, African American individuals from the same community are approximately twice as likely to develop Alzheimer disease. Despite this disparity, the largest Alzheimer disease genome-wide association studies to date have been conducted in non-Hispanic White individuals. In the largest association analyses of Alzheimer disease in African American individuals, ABCA7, TREM2, and an intergenic locus at 5q35 were previously implicated. Objective: To identify additional risk loci in African American individuals by increasing the sample size and using the African Genome Resource panel. Design, Setting, and Participants: This genome-wide association meta-analysis used case-control and family-based data sets from the Alzheimer Disease Genetics Consortium. There were multiple recruitment sites throughout the United States that included individuals with Alzheimer disease and controls of African American ancestry. Analysis began October 2018 and ended September 2019. Main Outcomes and Measures: Diagnosis of Alzheimer disease. Results: A total of 2784 individuals with Alzheimer disease (1944 female [69.8%]) and 5222 controls (3743 female [71.7%]) were analyzed (mean [SD] age at last evaluation, 74.2 [13.6] years). Associations with 4 novel common loci centered near the intracellular glycoprotein trafficking gene EDEM1 (3p26; P = 8.9 × 10-7), near the immune response gene ALCAM (3q13; P = 9.3 × 10-7), within GPC6 (13q31; P = 4.1 × 10-7), a gene critical for recruitment of glutamatergic receptors to the neuronal membrane, and within VRK3 (19q13.33; P = 3.5 × 10-7), a gene involved in glutamate neurotoxicity, were identified. In addition, several loci associated with rare variants, including a genome-wide significant intergenic locus near IGF1R at 15q26 (P = 1.7 × 10-9) and 6 additional loci with suggestive significance (P ≤ 5 × 10-7) such as API5 at 11p12 (P = 8.8 × 10-8) and RBFOX1 at 16p13 (P = 5.4 × 10-7) were identified. Gene expression data from brain tissue demonstrate association of ALCAM, ARAP1, GPC6, and RBFOX1 with brain ß-amyloid load. Of 25 known loci associated with Alzheimer disease in non-Hispanic White individuals, only APOE, ABCA7, TREM2, BIN1, CD2AP, FERMT2, and WWOX were implicated at a nominal significance level or stronger in African American individuals. Pathway analyses strongly support the notion that immunity, lipid processing, and intracellular trafficking pathways underlying Alzheimer disease in African American individuals overlap with those observed in non-Hispanic White individuals. A new pathway emerging from these analyses is the kidney system, suggesting a novel mechanism for Alzheimer disease that needs further exploration. Conclusions and Relevance: While the major pathways involved in Alzheimer disease etiology in African American individuals are similar to those in non-Hispanic White individuals, the disease-associated loci within these pathways differ.

Open-label randomized trial of torsemide compared with furosemide therapy for patients with heart failure.

PURPOSE: Because the bioavailability of oral furosemide is erratic and often incomplete, we tested the hypothesis that patients with heart failure who were treated with torsemide, a predictably absorbed diuretic, would have more favorable clinical outcomes than would those treated with furosemide. PATIENTS AND METHODS: We conducted an open-label trial of 234 patients with chronic heart failure (mean [+/- SD] age, 64 +/- 11 years) from an urban public health care system. Patients received oral torsemide (n = 113) or furosemide (n = 121) for 1 year. The primary endpoint was readmission to the hospital for heart failure. Secondary endpoints included readmission for all cardiovascular causes and for all causes, numbers of hospital days, and health-related quality of life. RESULTS: Compared with furosemide-treated patients, torsemide-treated patients were less likely to need readmission for heart failure (39 [32%] vs. 19 [17%], P <0.01) or for all cardiovascular causes (71 [59%] vs. 50 [44%], P = 0.03). There was no difference in the rate of admissions for all causes (92 [76%] vs. 80 [71%], P = 0.36). Patients treated with torsemide had significantly fewer hospital days for heart failure (106 vs. 296 days, P = 0.02). Improvements in dyspnea and fatigue scores from baseline were greater among patients treated with torsemide, but the differences were statistically significant only for fatigue scores at months 2, 8, and 12. CONCLUSIONS: Compared with furosemide-treated patients, torsemide-treated patients were less likely to be readmitted for heart failure and for all cardiovascular causes, and were less fatigued. If our results are confirmed by blinded trials, torsemide may be the preferred loop diuretic for patients with chronic heart failure.

Two-dimensional gel electrophoresis using the ISO-DALT system.

Two high-resolution electrophoretic procedures (isoelectric focusing and SDS-polyacrylamide gel electrophoresis) are combined in this unit to provide much greater resolution than either of the individual procedures. Solubilized proteins are first separated according to their isoelectric point by isoelectric focusing in a tube gel. This first dimension gel is then applied to the top of an SDS-polyacrylamide slab gel and electrophoresed. The proteins in the first-dimension gel migrate into the second-dimension gel where they are further separated on the basis of their molecular size. The ISO-DALT system was specifically designed for running multiple high-resolution two-dimensional gels at one time.

A comparison of aorta and vena cava medial message expression by cDNA array analysis identifies a set of 68 consistently differentially expressed genes, all in aortic media.

We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.

Plasminogen activator inhibitor type-1 and interleukin-6 in haemolytic uraemic syndrome.

OBJECTIVE: Because haemolytic uraemic syndrome (HUS) is an important cause of renal dysfunction in children, the availability of prognostic markers of disease severity could assist in identifying those at risk of developing long-term sequelae. The aim of this study was to test the hypothesis that plasma levels of plasminogen activator inhibitor type-1 (PAI-1) and interleukin-6 (IL-6) in children at the time of diagnosis of HUS would predict renal function outcome in terms of glomerular filtration rate (GFR). METHODOLOGY: Fourteen children suffering from diarrhoeal HUS were studied. Plasma samples were assayed for PAI-1 and IL-6, and GFR was measured at intervals after discharge from hospital. Twelve months following their recovery from HUS, the children were allocated to one of two outcome groups depending on whether GFR was above (Good Outcome, n = 9), or below (Poor Outcome, n = 5) 80 mL/min per 1.73 m2. RESULTS: Elevated concentrations of PAI-1 were found in 4 of 5 Poor Outcome and 4 of 9 Good Outcome children. At the same time, increased concentrations of IL-6 were observed in 3 of 5 Poor Outcome and 3 of 9 Good Outcome children. Renal function continued to be compromised in four Poor Outcome children 36 months after diagnosis. CONCLUSIONS: Our data show that PAI-1 and IL-6 are elevated in the plasma of some children at the time of diagnosis of HUS, but that neither is a definitive prognostic marker of poor outcome 3 years later.

Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells.

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.

CART peptides.

CART peptides are among the newest putative peptide neurotransmitter/cotransmitters. They show no significant homology to any other peptide, and they are thought to have a role in reward and reinforcement, feeding, development, sensory processing, stress and endocrine control.

CART: from gene to function.

CART was identified as a novel mRNA regulated by psychostimulant drugs. CART peptides appear to be neurotransmitters involved in a variety of functions such as feeding. The mouse gene has been characterized and localized to Chromosome 13. The processing of CART peptides is evident in Western blotting studies.

A systematic analysis of 40 random genes in cultured vascular smooth muscle subtypes reveals a heterogeneity of gene expression and identifies the tight junction gene zonula occludens 2 as a marker of epithelioid "pup" smooth muscle cells and a participant in carotid neointimal formation.

An accumulation of evidence suggests that vascular smooth muscle is composed of cell subpopulations with distinct patterns of gene expression. Much of this evidence has come from serendipitous discoveries of genes marking phenotypically distinct aortic cultures derived from 12-day-old and 3-month-old rats. To identify more systematic differences, we isolated 40 genes at random from libraries of these 2 cultures and examined message expression patterns. To determine consistency of differential expression, we measured mRNA levels in 4 sets of cultures in 6 phenotypically distinct aortic cell clones and in balloon injured rat carotid arteries to determine the relevance of these differences in vitro to in vivo biology. The following 5 consistently differentially expressed genes were identified in vitro: zonula occludens 2 (ZO-2); peroxisome proliferator-activated receptor delta (PPARdelta); secreted protein, acidic and rich in cysteine (SPARC); alpha1(I)collagen; and A2, an uncharacterized gene. We examined these 5 clones during carotid artery injury and an inconsistently differentially expressed clone Krox-24 because, as an early response transcription factor, it could be involved in the injury response. PPARdelta, A2, and Krox-24 mRNAs were upregulated during the day after injury. ZO-2 and alpha1(I)collagen messages were modulated for up to a month, whereas SPARC message showed no consistent change. An analysis of ZO-2 and other tight junction genes indicates that tight junctions may play a role in smooth muscle biology. These data suggest that a systematic analysis of these libraries is likely to identify a very large number of differentially expressed genes. ZO-2 is particularly intriguing both because of this tight junction gene's pattern of prolonged over-expression after injury and because of its potential role in determining the distinctive epithelioid phenotype of smooth muscle cells identified in rat and other species.

In vitro metabolism of ceftiofur in bovine tissues.

The metabolism of ceftiofur in bovine kidney, liver, muscle and lung, and the effects of the presence of cystine and glutathione in the media were evaluated using S-9 and microsomal tissue fractions. Conversion of ceftiofur to desfuroylceftiofur (DFC) was catalyzed by an esterase which was most active in kidney, followed by liver. It was not very active in muscle and lung. After DFC was liberated, it rapidly bound primarily to tissue proteins (> 56%), and was also conjugated to cysteine and glutathione. Production of DFC-cysteine by disulfide exchange of DFC with cystine and production of DFC-glutathione by conjugation of DFC to glutathione occurred in buffer if glutathione and cystine were present in the medium. These conjugations were also observed in incubations with tissue fractions, indicating that they were not inhibited by the tissues endogenous molecules. In addition, the metabolism of DFC-glutathione to DFC-cysteine was observed when tissue proteins were present. The metabolism of DFC-glutathione to DFC-cysteine was faster in kidney than in liver. Metabolites devoid of an intact beta-lactam ring were not observed in these in vitro studies.

Heterophilic NCAM-mediated cell adhesion to proteoglycans from chick embryonic brain membranes.

The vertebrate neural cell adhesion molecule NCAM mediates adhesion by both homophilic and heterophilic mechanisms, with heparan sulfate proteoglycans (HSPGs) being likely heterophilic ligands. In this study, transfected chicken NCAM polypeptides expressed on mouse L cells mediated the adhesion of these cells to several different heparan sulfate proteoglycans in nonionic detergent extracts of Embryonic Day 10 chicken brain membranes. In addition, adhesion inhibition experiments suggested a hitherto-undetected role for chondroitin sulfate proteoglycans in the stimulation of NCAM-mediated adhesion to some, but not all, of the HSPG ligands. Our experiments support the view that NCAM is a multivalent adhesive molecule whose function is affected by interactions with extracellular matrix and cell surface molecules.

The large cytoplasmic domain is not required for concentration of N-CAM at cell-cell contacts in transfected mouse neuroblastoma cells.

We examined the localization of the 140- and 180-kDa transmembrane isoforms of chicken N-CAM following transfection into mouse N2A neuroblastoma cells. Both isoforms were expressed at the cell surface and became partially or completely localized at areas of cell-cell contact after several days of culture or of in vitro differentiation. These results indicate that the presence of the large cytoplasmic domain of the 180-kDa N-CAM isoform is not necessary to bring about the localization of N-CAM to points of cell-cell contact.

Tissue distribution and cellular localization of hsp56, an FK506-binding protein. Characterization using a highly specific polyclonal antibody.

Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.

Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.

The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

HIV-1 protease cleaves actin during acute infection of human T-lymphocytes.

Actin, one of the most abundant proteins of the cell, is hydrolyzed by the human immunodeficiency virus type 1 (HIV-1) protease during acute infection of cultured human T lymphocytes. The actin fragments produced during the course of infection are identical to those obtained by recombinant HIV-1 protease digests of (1) a lysate from uninfected T lymphocytes and (2) globular actin itself. Hydrolysis by the HIV-1 protease of physiologically important host cellular proteins during infection may have important consequences relative to viral pathogenesis.

Tumor progression- and metastasis-associated proteins identified using a model of locally recurrent rat mammary adenocarcinomas.

A recently established model for local breast cancer recurrence using the 13762NF rat mammary adenocarcinoma was used to evaluate biologic and biochemical properties related to clinical outcome for this class of tumors. Sublines isolated from local tumor regrowths following surgical resection differed from each other and from the 'parental' cell lines for multiple phenotypes, including metastatic propensity. Local recurrence- and primary tumor-derived sublines were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), lectin binding to electrophoretically separated proteins, and lactoperoxidase-catalyzed cell surface iodination; and differential protein patterns were compared to tumor progression and metastatic potential. 2D-PAGE revealed several quantitatively different spots which correlated with lung colonization potential. In particular, quantities of an apparently unique, non-cell-surface protein, P50.9 (Mr approximately 50,900, pI approximately 7.3) correlated inversely with metastatic propensity, suggesting that it may be associated with, among other possibilities, the negative regulation of the metastatic phenotype. P50.9 was unrelated to four similarly sized metastasis-associated proteins--tumor autocrine motility factor; the rat analog of tumor suppressor, p53; rat cytokeratin 14 or procathepsin D--as determined by amino acid analysis. A major wheat germ agglutinin binding sialoglycoprotein, gp93 (Mr approximately 93,000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 greater than 'primary' tumor-derived lines (sc1, sc3) much greater than local recurrence-derived lines (LR1, LR1a, LR3, LR4, LR5, LR6)]. Besides cell surface glycoprotein losses, two of six local recurrence-derived sublines expressed a wheat germ agglutinin-binding sialoglycoprotein, gp110 (Mr approximately 110,000), previously undetected on any of the other cell lines including the parental populations. gp110 was found in LR3 and LR6 which were relatively highly metastatic; however, correlation with metastatic potential failed because gp110 was not present on the metastatic parental cell line, MTF7. These results demonstrate specific quantitative and qualitative protein differences associated with the selection of locally recurrent mammary tumors.

Effects of trospectomycin on serum sensitivity of Escherichia coli UC 9451.

Trospectomycin sulfate, a chemically synthesized analog of spectinomycin, exhibits a broad range of activity against both aerobes and anaerobes, including the etiological agents of sexually transmitted diseases. Its activity in vitro against Escherichia coli is considered only moderate. At subinhibitory levels, however, trospectomycin induced changes in a pathogenic strain of E. coli, UC 9451, which significantly increased its sensitivity to serum lysis. This strain of E. coli shows high-level resistance to serum in vitro, typically growing twofold within a 45-min incubation period. Following exposure to one-fifth the MIC of trospectomycin, greater than 99% of the bacteria were killed in 25% serum within 15 min. Surviving bacteria were static in this level of serum for over 3 h. Killing was due to lysis mediated by both the classical and alternative complement pathways. The bacteria exposed to trospectomycin were enlarged in both diameter and length, but they still grew at rates comparable to those of untreated bacteria. No other visible morphological changes could be directly related to the increase in serum sensitivity. The profile of outer membrane proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for trospectomycin-treated or untreated bacteria. However, the relative proportion of four major outer membrane proteins varied considerably.

Detection and recovery of proteins from gels following zinc chloride staining.

This report describes a method for detecting proteins in SDS polyacrylamide gels using ZnCl2 and their recovery using passive elution. Washing unfixed gels in a dilute solution of ZnCl2 produces two desirable effects. First, it makes the proteins easily visible as clear zones in a white background, and second, it prevents loss of proteins due to diffusion into the wash solution. Compared to other commonly used methods of protein detection such as Coomassie, KCl, copper or silver staining, the zinc stain offers some distinct advantages. Zinc staining can be completed in 15 min for most applications, making it much faster than Coomassie or most silver stains. The zinc stain is more sensitive than Coomassie, KCl or copper stain. Since no harsh chemical conditions are used in the zinc staining procedure, recovery of proteins from the gel is facilitated. More than 90% of selected proteins were recovered from 2-D gels by simple elution from the gel pieces with a buffer containing 10 mM EDTA.

Rapid modulation of a 64 K dalton fibroblast protein: a PDGF mediated early cellular event.

The results presented here reveal a novel platelet derived growth factor (PDGF) mediated early cellular event. Treatment of growth arrested Balb/c3T3 fibroblasts with PDGF induces a specific and rapid modulation of a 64,000 Dalton (64 KD) protein preexisting in quiescent cells. The kinetics of 64 KD protein modulation indicate that, temporally, this PDGF mediated step lies between the membrane associated immediate events such as receptor autophosphorylation or ion mobilization and the earliest known transcriptional event, the activation of the proto-oncogene c-fos.

Dietary management of canine and feline chronic renal failure.

Nutritional therapy is the mainstay of management of chronic renal failure in dogs and cats. Diets designed for use in renal failure are typically reduced in protein, phosphorus, and sodium content. These and other dietary modifications are designed to prevent or ameliorate clinical signs of uremia, minimize disturbances associated with excesses or losses of electrolytes and minerals, arrest or retard progression of renal failure, and maintain adequate nutrition.