Biomedical subjects

G Li

Publications and source records attributed to G Li.

At least 595 records · Page 33Linked to original sources

PubMed1998

p53-dependent DNA repair and apoptosis respond differently to high- and low-dose ultraviolet radiation.

p53 plays an essential part in the maintenance of the cellular genetic stability after a DNA-damaging event such as ultraviolet (UV) radiation. Following UV radiation, the amount of p53 protein is elevated. The increased p53 is believed to induce cell cycle arrest, promote nucleotide excision repair (NER) and apoptosis. To study if cells respond differently to high- and low-dose UV radiation, we examined the DNA repair efficiency and apoptosis rate of human and murine fibroblasts after UV radiation. Using a host cell reactivation assay, we found that NER was increased after low doses but not after high doses of UV radiation. In contrast, apoptosis occurred only after the cells received high doses (over 200 J/m2), but not low doses of UVB. The induction of both NER and apoptosis was observed only in p53+/+ murine fibroblasts, not in p53-/- cells, indicating that both stress response mechanisms are dependent on wild-type p53 function. UV radiation induced the expression of p53 protein in a dose-dependent manner up to 400 J/m2. In contrast, p21waf1/cip1 was induced only after low doses and bax only after high doses of UV radiation, supporting the roles of p21waf1/cip1 and bax in NER and apoptosis, respectively. Taken together, these results indicate that cellular stress response to UV radiation depends on UV dose, DNA repair after low doses and apoptosis after high doses, and that both mechanisms are dependent on wild-type p53 function.

Animals↗

PubMed1997

The Dictyostelium discoideum beta-1,4-mannosyltransferase gene, mntA, has two periods of developmental expression.

The precise roles of protein glycosylation in multicellular development are poorly understood. We have characterized the mntA gene from Dictyostelium discoideum which encodes the beta-1,4-mannosyltransferase enzyme that catalyzes the reaction: GDP-Man + dolichol-PP-GlcNAc2 --> dolichol-PP-GlcNAc2-Man + GDP. This gene has a central role in the synthesis of the lipid-linked oligosaccharide precursor which becomes the core of all asparagine-linked (N-linked) glycans. The mntA gene contains a single small intron and encodes a 493 aa protein with a predicted molecular size of 56 kDa. It is located 5' to the repE gene on chromosome IV and is transcribed in the opposite orientation to repE with which it shares a 585 bp of upstream intergenic region. The predicted mntA gene product shares 38% homology with the S. cerevisiae ALG1 gene product. The MntA protein has a region homologous to the putative dolichol-binding region in the yeast ALG1 protein, but it is located in a different part of the molecule. Northern analysis revealed that the expression of the mntA gene is regulated during multicellular development with two periods of mRNA accumulation. The mntA gene product has a classical endoplasmic reticulum retention motif, and is the first Dictyostelium gene encoding a protein that is active in this organelle. The identification of this gene will allow expanded studies of the role of N-linked glycans in multicellular development.

Amino Acid Sequence↗

PubMed1997

The DMB promoter: delineation, in vivo footprint, trans-activation, and trans-dominant suppression.

The HLA-DM loci encode the heterodimeric unconventional class II MHC molecules that are coexpressed with conventional class II MHC molecules. DM molecules are essential for the proper formation and function of conventional class II MHC molecules. This report characterizes the DMB promoter both by in vivo footprint and by in vitro functional analysis and reveals a promoter structure similar to that of conventional class II MHC genes. DR-negative mutant cell lines selectively defective in the transcription factor or class II trans-activator (CIITA) were used to reveal a requirement for both these factors in DMB promoter activation. Complementation of defective cell lines with the appropriate transcription factor reconstituted DMB promoter activation. Further analysis with CIITA identified several mutant forms of CIITA that are trans-dominant-negative mutants, i.e., they suppressed DMB promoter activation by transfected and endogenous CIITA. These mutants may be used in abiological setting to down-regulate the function of DM in Ag processing.

Base Sequence↗

PubMed1997

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

ADP Ribose Transferases↗

PubMed1997

Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue.

BACKGROUND: Fetal rat cardiomyocytes transplanted into left ventricular scar tissue of the adult rat heart limit scar expansion and improve heart function. This study determined morphologic changes of transplanted fetal rat cardiomyocytes in myocardial scar tissue. METHODS AND RESULTS: The left ventricles of 500-g Sprague-Dawley rats were cryodamaged. At 4 weeks after myocardial injury, a transmural scar (54+/-11 mm2) (mean+/-1 SDak) formed at the apex (n=6). Cardiomyocytes freshly isolated from 18-day-gestation Sprague-Dawley rat hearts were transfected with plasmid containing the beta-galactosidase and then injected into the 4-week-old scar tissue. Cell culture medium was injected into the scar tissue of control animals. At 4 weeks posttransplantation, the cardiomyocytes had formed cardiac tissue (20.7+/-6.9 mm2, n=14), which stained positively for beta-galactosidase activity in the scar (90.4+/-25 mm2, n=14). The transplanted cardiomyocytes formed sarcomeres and were linked by junctions composed of desmosomes and fascia adherens. Lymphocyte infiltration occurred despite use of cyclosporin A. No myocardial tissue was found in the scar tissue of the control animals (n=14). More arterioles and venules were found (P<.01) in the cardiomyocyte grafts (1.2+/-0.6 vessel/0.8 mm2; n=14) than in the control scar tissue (0.1+/-0.1 vessels/0.8 mm2; n=14). At 20 weeks post-transplantation, the transplant tissue size (6+/-6 mm2; n=7) was smaller (P=.007) than 4-week old transplant, and the scar (162+/-46 mm2; n=7) was larger (P=.005) than 4-week-old scar. Lymphocyte infiltration was still present among the remaining transplanted cells. CONCLUSIONS: This study demonstrated that cardiac tissue formed by transplanted fetal cardiomyocytes in the myocardial scar tissue decreased in size with time probably secondary to rejection.

Animals↗

PubMed1997

The serine chemoreceptor from Escherichia coli is methylated through an inter-dimer process.

Covalent modification of receptors is a widespread phenomenon in signal transduction. In the chemosensory system of Escherichia coli, the reversible methylation of certain glutamic acid residues in the cytoplasmic domain of receptor homodimers mediates adaptation to stimuli. Here we report that the serine receptor is methylated by an inter-dimer process. Methyltransferase bound to one subunit in a serine receptor homodimer was found to catalyze the addition of methyl groups to a receptor subunit in an adjacent dimer in the membrane. These results demonstrate a role for inter-dimer interactions in transmembrane signaling.

Bacterial Proteins↗

PubMed1997

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.

3-O-Methylglucose↗

PubMed1997

Activation and transdominant suppression of MHC class II and HLA-DMB promoters by a series of C-terminal class II transactivator deletion mutants.

The class II transactivator (CIITA) is a highly specific transcription factor that activates only genes known to be involved in the class II MHC processing pathway, including class II MHC, invariant chain, and HLA-DMA/B genes. In this work, we show the requirement of a new region in CIITA that is critical for its function. Deletion mutants lacking varying length of the C terminus show that the C-terminal 41 amino acids of CIITA are indispensable for the activation of both the conventional DRA and nonconventional DMB promoters. This region contains a highly charged stretch of amino acids that is homologous to a yeast transcription factor, repression activator protein-1 (RAP1)-interacting factor 1. Mutants lacking the C terminus were tested in a transdominant-negative assay to examine their capacity to block the wild-type CIITA function. Two of these deletion mutants suppressed the activity of endogenously expressed wild-type CIITA to activate both DRA and DMB promoters. It may be possible to utilize these mutants to modulate MHC class II and DM gene expression.

Amino Acid Sequence↗

PubMed1997

Synthesis and properties of second-generation 2-5A-antisense chimeras with enhanced resistance to exonucleases.

In order to stabilize 2-5A-antisense chimeras to exonucleases, we have synthesized chimeric oligonucleotides in which the last phosphodiester bond at the 3'-terminus of the antisense domain was inverted from the usual 3',5'-linkage to a 3',3'-linkage. The preparation of such analogues was accomplished through standard phosphoramidite chemistry with the use of a controlled pore glass solid support with a nucleoside attached through its 5'-hydroxyl, thereby permitting elongation at the 3'-hydroxyl. The structures of such terminally inverted linkage chimeras of the general formula pA4-[pBu]2-(pdNn3'-3'dN) were corroborated by a combination of snake venom phosphodiesterase digestion in the presence or absence of bacterial alkaline phosphatase. Most characteristically, the presence of the 3'-terminal-inverted phosphodiester linkage produced an unnatural dinucleotide of general composition dN3'p3'dM. These structures could be confirmed by independent synthesis and fast atom bombardment mass spectroscopy (FAB). 2-5A-Antisense chimeras of this structural class, pA4-[pBu]2-(pdNn'3-3'dN), were 5-6-fold more stable than their unmodified congeners, pA4-[pBu]2-(pdN)n, to degradation by a representative phosphodiesterase from snake venom. In 10% human serum, the new 2-5A-antisense chimeras, pA4-[pBu]2-(pdNn3'-3'dN), possessed a half-life that was 28-fold longer than that of the unmodified chimeras. These results provide entry to a second generation of 2-5A-antisense chimeras.

Adenine Nucleotides↗

PubMed1997

Interactions between smooth muscle alpha-actinin and lipid bilayers.

alpha-Actinin has been proposed to be the actin-plasma membrane linker. This assumption is based on the discovery of direct interaction of alpha-actinin with two specific lipids, diacylglycerol and palmitic acid [Burn, P. (1988) Trends Biochem. Sci. 13, 79-83]. In our study, the binding of alpha-actinin with vesicles containing negatively charged phospholipids was measured by the method of 90 degrees light-scattering. Our results show that alpha-actinin is able to bind membranes containing negatively charged phospholipids, but not to bind membranes composed of neutral lipids only. Diacylglycerol and palmitic acid, on the other hand, have little effect on the binding of alpha-actinin to lipid vesicles. Analysis of binding isotherms in terms of a membrane binding model gave apparent dissociation constants which varied between 0.2 and 3 microM over a range of 5-20 mol % negatively charged phospholipid. Comparing the kinetics of alpha-chymotrypsin digestion of alpha-actinin in solution to those of vesicle-bound alpha-actinin, it can be seen that the cleavage site at the junction between the C-terminal and the central rod domain of alpha-actinin and another cleavage site on the C-terminal domain can be most effectively protected by its membrane binding. Analysis of the amide I and II regions of Fourier-transform infrared spectra of alpha-actinin revealed that the association of alpha-actinin with negatively charged phospholipid vesicles resulted in some perturbation of the protein secondary structure. Monolayers containing negatively charged phospholipid were layered and incubated on the surface of a polymerization solution of actin and alpha-actinin, and observed with an electron microscope. The results show that the bundle structure of actin filaments can be formed if diacylglycerol and palmitic acid are present in lipid layers.

Actinin↗

PubMed1997

Overexpression of transforming growth factor-beta1 and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy.

BACKGROUND: Idiopathic hypertrophic cardiomyopathy (HCM) is characterized by regional myocardial hypertrophy. To investigate involvement of growth factors on myocardial hypertrophy in HCM patients, we evaluated gene expression and cellular localization of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factors (IGF-I and IGF-II), and platelet-derived growth factor-B (PDGF-B) in ventricular biopsies obtained from patients with HCM (n=8), aortic stenosis (AS) (n=8), or stable angina (SA) (n=8) and from explanted hearts with ischemic cardiomyopathy (TM) (n=7). METHODS AND RESULTS: Levels of TGF-beta1, IGF-I, IGF-II, and PDGF-B transcripts were quantified with the use of multiplex RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal standard. Antibodies against TGF-beta and IGF-I were used to localize their peptides within the myocardium. Antisense and sense (control) cRNA probes of TGF-beta1 and IGF-I, labeled with digoxigenin, were used to localize the growth factor transcripts by in situ hybridization. mRNA levels (densitometric ratio of growth factor/glyceraldehyde-3-phosphate dehydrogenase) of TGF-beta1 and IGF-I in HCM (0.75+/-0.05 and 0.85+/-0.15, respectively; mean+/-1 SEM) were significantly (P<.01 for all groups) elevated in comparison with non-HCM myocardium (AS: 0.38+/-0.07, 0.29+/-0.06; SA: 0.32+/-0.04, 0.18+/-0.05; TM: 0.25+/-0.03, 0.15+/-0.03). mRNA levels of TGF-beta1 and IGF-I in the hypertrophic AS myocardium were greater (P=.02, P=.05) than those in the explanted myocardium (TM). Immunohistochemical and in situ hybridization studies showed increased expression of TGF-beta1 and IGF-I in the HCM cardiomyocytes. CONCLUSIONS: Gene expression of TGF-beta1 and IGF-I was enhanced in idiopathic hypertrophic cardiomyopathy and may be associated with its development.

Aged↗

PubMed1997

Inhibition of calcium-induced insulin secretion from intact HIT-T15 or INS-1 beta cells by GTP depletion.

Using intact rat islets, we previously observed that GTP depletion (achieved through the use of mycophenolic acid or other synthesis inhibitors) impedes nutrient- but not K+-induced insulin secretion. It was concluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca2+-induced exocytosis itself) is modulated by ambient GTP levels. To examine Ca2+-dependent steps further in intact beta cells, INS-1 cells (which synthesize GTP and ATP similarly to rat islets) and HIT-T15 cells (whose synthesis of purine nucleotides is different) were studied following cell culture for 1-18 hr in various concentrations of mycophenolic acid (MPA) or mizoribine (MZ). Both agents profoundly reduced GTP content (mean: -78%) and lowered the GTP/GDP ratio by an average of -73%; concomitantly, MPA or MZ reduced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 100 microM tolbutamide, independent of any changes in cell viability, insulin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca2+ concentrations. In INS-1 cells (which appear to have normal nucleobase transport and "salvage" pathway activities), guanine (but not adenine) restored GTP content, the GTP/GDP ratio, and Ca2+-induced secretion. In HIT cells, the phosphoribosylation of exogenous guanine or hypoxanthine is defective; however, provision of 500 microM guanosine (but not adenosine) reversed the effects of MPA. We conclude that, at least in certain situations, a requisite role for GTP in the distal step(s) of exocytosis can be demonstrated.

Animals↗

PubMed1997

Validity of the family history method for identifying schizophrenia-related disorders.

We examined the family history method's validity for identifying schizophrenia related disorders (SRD) by comparing family history and family study derived diagnoses. First degree relatives (n = 284) of 48 psychiatrically disordered probands, predominantly with schizophrenia, were diagnosed using the Family History RDC (FH-RDC) which include three psychotic schizophrenia related disorders (P-SRD): schizophrenia, chronic SAD and chronic unspecified functional psychosis (CUFP). Supplementary criteria for schizophrenia related personality disorders (SRP), derived to identify schizotypal and paranoid personality disorders (PD), were also assessed. About two thirds of these relatives (n = 196; 69.0%) were independently diagnosed by RDC and DSM-III-R on both axis I and axis II in a family study. The specificity was 1.0 (178/178) and the sensitivity of the family history derived diagnosis for P-SRD was 0.72 (13/18). Sensitivity for P-SRD was improved, however, by inclusion of SRP which captured three of the five false negative relatives. The sensitivity of SRP for schizotypal or paranoid PD was 0.39 (15/38) and the specificity was 0.92 (127/138). The FH-RDC have moderately good sensitivity and excellent specificity for the psychotic schizophrenia related disorders. While family history criteria for SRP are not a good proxy for schizotypal or paranoid PD, they can enhance the family history method's sensitivity for SRD.

Adult↗

PubMed1997

Uncoupling of membrane ruffling and pinocytosis during Ras signal transduction.

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling.

Androstadienes↗

PubMed1997

Correlation of selective modifications to a 2',5'-oligoadenylate-3',5'-deoxyribonucleotide antisense chimera with affinity for the target nucleic acid and with ability to activate RNase L.

The use of an antisense oligonucleotide to address a specific targeted RNA sequence and subsequent localized activation of the 2-5A-dependent RNase (RNase L) to effect selective RNA degradation is a new approach to the control of gene expression called 2-5A-antisense. The previously reported biological activity of the 2-5A:AS chimeric oligonucleotide [p5'(A2'p)3A-antiPKR1], directed against nucleotides 55-73 of the coding sequence of the PKR mRNA, has been used as a point of reference to examine the effect of introducing mismatches into the chimeric oligonucleotide, altering the chain length of the antisense domain of the chimeras, removal of the 5'-monophosphate moiety, shortening the 2',5'-oligoadenylate domain, and substitution of 3',5'-linked 2'-deoxyadenosine nucleotides for the 2-5A domain. The general formula for the novel chimeric oligonucleotides is p5'(A2'p)3A2'p(CH2)4p(CH2)4p(5'N3'p)mN, where N is any nucleoside and m is any integer. When the biological activity of these new chimeric oligonucleotides was compared to that of the parent chimera, 2-5A-aPKR, for their ability to effect target PKR RNA cleavage in a cell-free and in an intact cell assay, it was determined that there was a close correlation between the activity of 2-5A-antisense chimeras and their affinity (Tm) for a targeted nucleic acid. In addition, there was also a close correlation between activity of the 2-5A-antisense chimeras and their ability to activate the 2-5A-dependent RNase L.

Adenine Nucleotides↗

PubMed1997

A novel hamster prion protein mRNA contains an extra exon: increased expression in scrapie.

Prion protein (PrP) is the only known constituent of the agents (called prions) that cause fatal neurodegenerative diseases in animals and humans. PrP derives from a host protein encoded by a single copy gene having three known exons in mice, cattle and sheep but only two exons in hamsters and humans. We have identified and sequenced the missing exon from the hamster PrP gene. The new hamster PrP exon is 83% identical to mouse exon 2 and 76% identical to exon 2 from cattle and sheep. PrP mRNAs containing the new exon 2 (mRNA[1+2+3]) were expressed in the colliculi, frontal cortex and hippocampus of normal hamsters at approximately 30% to approximately 50% of the levels of the mRNA without exon 2 (mRNA[1+3]). Expression of PrP mRNA[1+2+3] was increased in the colliculi beginning 49 days after inoculation with scrapie prions and reached a level 2.5 times normal by day 77. Increased expression of PrP mRNA[1+2+3] in the colliculi correlated with expression of glial fibrillary acidic protein (GFAP) mRNA. Expression of GFAP and PrP mRNAs was not significantly increased in the hippocampus or the frontal cortex during the disease. Our study shows that exon 2 plays a role in regulating the cellular expression of hamster PrP and suggests that mRNA[1+2+3] may be preferentially expressed in hamster astrocytes.

Animals↗

PubMed1997

Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells.

Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.

Adenine Nucleotides↗

PubMed1997

The validity of the family history method for identifying Alzheimer disease.

OBJECTIVE: To examine the validity of the family history method for identifying Alzheimer disease (AD) by comparing family history and neuropathological diagnoses. METHODS: Seventy-seven former residents of the Jewish Home and Hospital for the Aged, New York, NY, with neuropathological evaluations on record were blindly assessed for the presence of dementia and, if present, the type of dementia through family informants by telephone interviews. The Alzheimer's Disease Risk Questionnaire was used to collect demographic information and screen for possible dementia. If dementia was suspected, the Dementia Questionnaire was administered to assess the course and type of dementia, i.e., primary progressive dementia (PPD, likely AD), multiple infarct dementia, mixed dementia (i.e., PPD and multiple infarct dementia), and other dementias based on the modified Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria. RESULTS: Sixty (77.9%) of 77 elderly subjects were classified as having dementia and 17 (22.1%) were without dementia by family history evaluation. Of the 60 elderly subjects with dementia, 57 (95%) were found at autopsy to have had neuropathological changes related to dementia. The sensitivity of the family history diagnosis for dementia with related neuropathological change was 0.84 (57 of 68) and the specificity was 0.67 (6 of 9). Using family history information to differentiate the type of dementia, the sensitivity for definite or probable AD (with or without another condition) was 0.69 (36 of 51) and the specificity was 0.73 (19 of 26). The majority (9 of 15) of patients testing false negative for PPD had a history of stroke associated with onset of memory changes, excluding a diagnosis of PPD. CONCLUSIONS: Identifying dementia, in general, and AD, in particular, has an acceptable sensitivity and specificity. As is true for direct clinical diagnosis, the major issue associated with misclassifying AD in a family history assessment is the masking effects of a coexisting non-AD dementia or dementia-related disorders, such as stroke. Including mixed cases, ie, PPD and multiple infarct dementia in estimates of the familial risk for AD can reduce the extent of underestimation of PPD.

Aged↗