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.

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.

Mode of fibroblast growth enhancement by human interleukin-1.

Previous studies have demonstrated that interleukin-1 (IL-1) stimulates fibroblast growth (Schmidt, J. A., S. B. Mizel, D. Cohn, and I. Green. 1982. J. Immunol. 128:2177-2182) and binds to specific, high affinity receptors of BALB/c3T3 cells (Bird, T. A., and J. Saklatval. 1986. Nature (Lond.). 324:263-265, 266-268). We have investigated the mechanism of fibroblast growth stimulation by IL-1. Addition of fibroblast growth factor derived from platelets (PDGF) to a quiescent culture of BALB/c3T3 cells produced 8-10-fold increase in DNA synthesis during 24-h incubation. The cellular action of PDGF was mediated through competence induction and required synergistic action of plasma-derived factors for full mitogenic activity. When tested at a wide range of concentrations (0.1-100 pM), natural IL-1 or recombinant IL-1 produced only a maximum of 5-10% of DNA synthesis elicited in response to PDGF or serum. Induction of DNA synthesis required continuous presence of IL-1 and did not exhibit synergism with plasma. Competence induction and mitogenic stimulation by PDGF was associated with early induction of proteins P32, P38, P46-48, P75, and changes in cytoskeletal organization. Examination of these early cellular changes showed that IL-1 did not produce similar induction of cellular proteins and the morphological changes associated with growth stimulation. These results suggest that the mode of IL-1 action on BALB/c3T3 was not through competence induction. When IL-1 was added to cells rendered competent by brief exposure to PDGF, 10-15% additional DNA synthesis occurred during the first 24 h. Extended incubation of PDGF-treated cells in the presence of IL-1 revealed that the stimulation by IL-1 occurred predominantly during the subsequent cycle of DNA replication, wherein DNA synthesis reached three- to fivefold higher than the untreated cultures. We conclude (a) IL-1 alone is not a potent mitogen for BALB/c3T3 cells, and does not bring cells out of the growth arrest Go phase, (b) treatment with PDGF renders the cells more responsive to IL-1, (c) part of the IL-1 action on competent cells may be characterized as progression inducing activity, further, (d) our results indicate that action of IL-1 on PDGF-treated cells produces sustained DNA synthesis for an extended period, perhaps by preventing the entry of cells into growth arrest Go phase.

Increased amount of a 25-kilodalton phosphoprotein after v-mos transfection of CHO cells.

We transfected Chinese hamster ovary (CHO) cells with a cloned v-mos gene (pHT25). The mos family of oncogenes has previously been shown to have serine-threonine kinase activity. This kinase activity may be required for oncogenic transformation, although its exact biological role is unknown. We found that the transfected cells had an altered morphology, a slower doubling time, and an apparent increase in the amount of a 25-kilodalton (kDa) phosphoprotein that appeared to be of low abundance. Transfection of CHO cells with a cloned temperature-sensitive mos gene (ts159) led to isolation of a cell line that showed the presence of the 25-kDa phosphoprotein at the permissive but not at the nonpermissive temperature, suggesting a direct relationship between mos activity and the presence of this phosphoprotein. The characteristics of altered morphology and depressed growth rate were reminiscent of changes seen after the activation of the cyclic AMP-dependent protein kinase (PKA) in CHO cells. However, PKA activation did not stimulate phosphorylation of this 25-kDa protein, nor was there a change in total PKA activity in these cells. We suggest that the increased presence of the 25-kDa phosphoprotein is a consequence of the v-mos transfection and that it may be involved in the change of morphology and growth rate seen in the CHO cells. Phosphorylation of this protein may be a useful marker of mos and have some functional importance in the transformation of cells by the v-mos oncogene.

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.

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.

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.

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.

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.

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.

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.

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.

Measuring various drug use dimensions with a calendar method.

This note presents a technique used to acquire a variety of time-specific data on drug use. The calendar technique involves self-reported patterns of consumption and any changes therein, including cessation. Advantages of this technique over other approaches are discussed, and examples of the variety of use measures that can be derived from the calendar method are illustrated.

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.

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.

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.

Insulin-induced rapid decrease of a major protein in fat cell plasma membranes.

In order to increase our understanding of the mode of action of insulin in rat fat cells, we investigated the effect of insulin on protein concentrations in purified fat cell fractions using two-dimensional electrophoresis in combination with an ultrasensitive color silver stain technique. Incubation of fat cells with insulin caused a 90% decrease in the plasma membrane concentration of a major plasma membrane protein with a molecular mass of 90 kDa. The insulin effect was dose-dependent with a half-maximal effect at 9.5 microunits/ml, and time-dependent with a t 1/2 of less than 20 s. Insulin-like growth factor I, orthovanadate, and lanthanum mimicked the effect of insulin. Likewise, fractionation of adipocytes in the presence of divalent cation chelating agents caused a similar reduction in the concentration of the 90 kDa protein, and it was possible to overcome the effects of the chelating agents by adding equivalent amounts of calcium. This suggests the involvement of calcium. The 90 kDa protein was also found in low and high density microsomes, but it was not affected in those fractions by either insulin or chelators. It is suggested from the study that the movement of a 90 kDa protein in fat cell plasma membranes probably represents part of the transmission system in the mechanism of insulin action in rat adipocytes.

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.

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.