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Mutation-selection balance at a modifier-of-imprinting locus.

We propose a pair of population genetic models for a modifier-of-imprinting locus for which different genotypes imprint different proportions of an imprintable target locus in their gametes. The two models examine the situations in which imprinting is advantageous, and we discuss three cases for which the modifier is respectively partially dominant, dominant, or recessive. The models predict the stable equilibrium frequencies of the mutant modifier and functionally diploid individuals in a large population in terms of up to four parameters: the mutation rate at the modifier locus, nu; the selection coefficient against the disadvantageous phenotype, sigma; the proportion of unimprinted eggs produced by homozygotes for the mutant modifier, theta, and, in the partially dominant models, the dominance parameter, kappa. The equilibrium frequency of the mutant phenotypes is shown to be approximately twice that of standard Mendelian models: 2 nu/sigma or 4nu/sigma when the modifier is recessive or dominant, respectively. Mathematical equivalences between these and nonimprinting models are noted.

Chromosome Mapping

Efficacy of modified human immune serum globulin in the treatment of experimental murine infections with seven immunotypes of Pseudomonas aeruginosa.

Modified immune serum globulin, prepared from human immune serum globulin by a nonenzymatic method, is apparently safe for intravenous administration to humans. The efficacy of the preparation was determined in experimental murine infections with seven immunotypes of Pseudomonas aeruginosa. Intravenously administered 0.85 percent NaCl, 0.3 m glycine, and 10 percent human albumin did not protect against lethal pseudomonas infection, whereas modified immune serum globulin given by the same route did protect mice. In the mouse protection test, the mean dose of the preparation that saved 50 percent of mice infected with any of eight strains of P. aeruginosa was 480 mg/kg (range, 12-2,333 mg/kg). For five strains the 50 percent effective dose was smaller than 200 mg/kg. There was no correlation between the efficacy of modified immune serum globulin in the mouse protection test and titers of antibody, as determined by bacterial agglutination. Therapy of pseudomonas infection in mice with modified immune serum globulin was followed by a prompt and persistent decrease in the numbers of intraperitoneal bacteria. This finding is consistent with the interpretation that modified immune serum globulin acts primarily as an opsonin and not as an antitoxin. Modified immune serum globulin may prove to be useful in the treatment of human infections.

Agglutination Tests

Z-DNA conformation of N-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) determined by reactivity with anti cytidine antibodies and minimized potential energy calculations.

The conformation of poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), and calf thymus DNA modified with N-acetoxy-N-2-acetylaminofluorene (N-acetoxy-AAF) was examined by extent of reaction with anti cytidine antibodies. In contrast to modified poly(dG).poly(dC0 and DNA, modified poly(dG-dC).poly (dG-dC) failed to react with the antibodies indicating that the base pairing in this polymer is intact. This in consistent with induction of the Z-DNA conformation in AAF modified poly(dG-dC).poly(dG-dC). Using minimized potential energy calculations on the dCpdG-AAF dimer as a model for the modified polymer, it is shown that the proposed Z-DNA conformation is energetically stable. A model is proposed for an AAF modified tetramer, dGpdCpdGpdC, in which the AAF is external to the Z-DNA duplex.

2-Acetylaminofluorene

Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. II. Identification of the histidine residue of beta1-bungarotoxin modified by p-bromophenacyl bromide.

beta1-Bungarotoxin modified with p-bromophenacyl bromide (BPB) was reduced and carboxymethylated, and the resulting two constituent RCM-polypeptide chains (the RCM-A and B chains) were separated. The RCM-A chain was found to be modified by BPB by measuring its UV absorption spectrum and was shown to have lost one histidine residue by analyzing its amino acid composition. To determine the location of the modified histidine residue in the A chain of the toxin, the RCM-A chain was digested with TPCK-trypsin, and the resulting peptides were fractionated by gel filtration followed by DEAE-cellulose chromatography. The modified residue was finally identified as histidine-48 in the A chain by Edman degradation and from the amino acid composition of the BPB-modified peptide. The amino acid sequence around the modified histidine residue in the A chain is highly homologous with those of porcine pancreas phospholipase A2 and presynaptic toxin, notexin. We conclude that histidine-48 in the A chain participates in the phospholipase A activity of beta1-bungarotoxin.

Acetophenones

Bindings of Ca2+ and substrate analogs to a cobra venom phospholipase A2 in which the alpha-amino group is modified to an alpha-keto group.

The pH dependence of the chemical reaction rate of p-bromophenacyl bromide (BPB) with His 48 of cobra (Naja naja atra) venom phospholipase A2, in which the alpha-NH2 group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 in the absence of Ca2+. The pH-dependence curve was monophasic with a midpoint at pH 7.9, which corresponds to the pK value of His 48 of the alpha-NH2-modified enzyme, whereas the curve for the intact enzyme was biphasic, indicating participation of two ionizable groups with pK values of 7.3 and 8.55 (Teshima et al. (1982) J. Biochem. 91, 1778-1788). These two groups were thus identified as His 48 and the alpha-NH2 group, respectively. The pH dependence of the binding constant of Ca2+ to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by measuring the tryptophyl fluorescence changes. The pH-dependence curve was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 13-20), and it was interpreted in terms of participation of His 48 and Asp 49 (pK 5.4). The absence of participation of the alpha-NH2 group in the Ca2+ binding was thus confirmed. Bindings of monodispersed n-dodecylphosphorylcholine (n-C12PC) and micellar n-hexadecylphosphorylcholine (n-C16PC) to the alpha-NH2-modified enzyme were studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) and tryptophyl fluorescence methods, respectively. The binding constant of the monodispersed substrate was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The binding constant of the micellar substrate to the modified enzyme in the presence of Ca2+ was also very similar to that for the intact enzyme-Ca2+ complex (Teshima et al. (1983) J. Biochem. 94, 223-232), and the pH-dependence curve was interpreted in terms of participation of His 48. On the other hand, the binding constant of the micellar substrate to the modified apoenzyme was much smaller than that for the intact apoenzyme. Nevertheless, the pH-dependence curve could be interpreted in terms of participation of His 48 and Asp 49. From these findings, it was concluded that the ionization state of the alpha-NH2 group of cobra venom phospholipase A2 is essentially irrelevant to the bindings of Ca2+ and also of the monodispersed and micellar substrates.

Animals

Modified essay question.

This article examines the reliability and validity of the modified essay question. An 18-item modified essay question was developed and pretested on a group of clinical experts. After the pretest, the modified essay question was administered to a group of 25 physical therapy students immediately before their second level orthopedic placement. A reliability index of .39 (coefficient alpha) was calculated for the test. In addition, measures of validity were obtained by correlating the modified essay question scores with multiple-choice question scores and the students' in-clinic reasoning scores. The results suggest that the reliability coefficient found in this study is comparable to the medical literature. Also, the results showed that the modified essay question demonstrated a greater correlation with the in-clinic clinical reasoning score compared with the parallel content multiple-choice examination. Furthermore, preliminary evidence suggests that the modified essay question may be a useful paper and pencil tool in evaluating clinical reasoning. Finally, several shortcomings of this study are discussed in conjunction with a direction for further study.

Adult

The generation of cytotoxic T lymphocytes against acetaldehyde-modified syngeneic cells.

The major metabolic product of ethanol is acetaldehyde. It is highly reactive with proteins. In situ this modification is significant enough to generate an antibody response. Whether an effector cellular immune response can be generated against these acetaldehyde modified adducts on syngeneic cells is not known. In this paper we have demonstrated in the murine system that acetaldehyde modified splenic cells can generate cytotoxic T lymphocytes (CTL). These CTL are specific for the acetaldehyde modified syngeneic cells, and not acetaldehyde modified allogeneic cells. The ability of the CTL to lyse-specific targets is dependent on the formation of stable acetaldehyde adducts. Cold target inhibition studies reveal that modified syngeneic cells can inhibit lysis as effectively as unmodified cells. Therefore, the present study lends support to the hypothesis that acetaldehyde modified cells can generate a cellular immune response and may do so in pathologic states.

Acetaldehyde

Relationship between alcohol intake and immunoglobulin a immunoreactivity with acetaldehyde-modified bovine serum albumin.

Acetaldehyde, the main metabolite of ethanol, is a highly reactive species that reacts with macromolecules to produce unstable and stable adducts. Acetaldehyde-modified proteins are immunogenic and have been detected in the liver and blood of alcoholics. Furthermore, antibodies reactive with acetaldehyde-modified proteins have been detected in the plasma of social drinkers and alcoholics. However, the class distribution of immunoglobulins reactive with modified proteins was different in the two groups, being predominantly immunoglobulin (Ig)M in social drinkers, but IgM and IgA in alcoholics. In this study, we demonstrate that heavy drinkers (alcohol intake > 130 g/week for females and 150 g/week for males) also exhibit IgA reactivity with acetaldehyde-modified proteins. The IgA adduct-specific reactivity (IgA reactivity with acetaldehyde-modified bovine serum albumin-reactivity with native bovine serum albumin) showed a moderate correlation with self-reported alcohol intake, but did not correlate with markers such as plasma transaminase, gamma-glutamyltransferase activity, or mean corpuscular volume. IgA adduct-specific reactivity had similar specificity to the conventional tests of alcohol abuse, but had higher sensitivity than the other tests, especially with heavy drinkers. Data presented herein demonstrate that elevated IgA reactivity with acetaldehyde-modified epitopes is associated with heavy drinking and is a potential marker for high alcohol intake.

Acetaldehyde

Physiological responses of horses competing at a modified 1 star 3-day-event.

The impending 1996 summer Olympic 3-day-event in Atlanta has focused attention on the need to determine what modifications to the demanding Endurance Test will be required to ensure safety of the horses competing. Three groups of horses participated in a Field Trial held in August of 1994 in northern Georgia to determine the safety and feasibility of conducting a modified 3-day-event in hot, humid weather. One group (TD) completed a modified 1 Star 3-day-event test, a control group (HT) completed a Horse Trial identical to the modified 1 Star test except for the omission of Phases B and C and the third group (E), comprised of European horses, completed the modified 1 Star test with a longer, faster Phase C than was used for TD. During the Endurance Test, the ambient temperature and relative humidity ranged from 24.3 degrees C and 98.9% in the morning to 30.2 degrees C and 51.6% in the afternoon. No horse failed to complete the Trial because of heat stress or fatigue. There were no significant (P < 0.05) differences detected in heart rate, rectal temperature, respiratory rate or net weight loss between HT and TD horses at any observation time. The highest rectal temperature recorded at the end of Phase C was 39.6 degrees C. These findings suggest that the modified 1 Star Endurance Test was as well tolerated by American horses as the control Horse Trial test. Rectal temperature was significantly higher for E than for TD or HT at the finish of Phase C. European horses had significantly greater decreases in weight than HT and TD at the end of Phases C and D and the next day. These findings probably reflect the faster and longer work effort of E horses during Phase C. Modification of Phase C and the rest-pause to ensure that recovery and heat dissipation occurred before the start of Phase D resulted in a 3-day-event that was safe for horses. The Field Trial provides a model for designing a modified Olympic Endurance Test. If the 1996 Olympic 3-day-event is held in hotter and more humid weather than the Field Trial, additional modifications to the Endurance Test (decreased distances, speeds and numbers of jumping efforts) will probably be required to ensure safety of competing horses.

Animals

Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus.

In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.

Amino Acid Sequence

Immunological detection of the Kanagawa phenomenon of vibrio parahaemolyticus on modified selective media.

Selective media for Vibrio parahaemolyticus, BTB-Teepol agar and modified arabinose-ammonium sulfate-cholate agar, were modified for use in immunological detection of the thermostable direct hemolysin produced by this organism. The modified BTB-Teepol agar and modified arabinose-ammonium sulfate-cholate agar were both found to be useful for the modified Elek test and immunohalo test with antiserum (or immunoglobulin G) against the thermostable direct hemolysin. With these modified media it is possible to isolate V. parahaemolyticus and identify the Kanagawa phenomenon on a single plate and thus save time in obtaining results.

Culture Media

Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy.

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.

AIDS-Related Opportunistic Infections

Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins.

Antibodies to polyethylene glycol (PEG) were raised in rabbits by immunization with monomethoxy polyethylene glycol modified ovalbumin (OA), bovine superoxide dismutase (SOD), and ragweed pollen extract (Rag), given in Freund's complete adjuvant (FCA). Immunogenicity depended on the nature of the protein and the degree of modification. With modified OA, in the presence of FCA, the majority of animals showed an anti-PEG response. With modified SOD and Rag only a small proportion of animals responded. In the absence of FCA, modified OA, given s.c., did not elicit any anti-PEG antibody response in rabbits and only a weak response in mice. PEG of MW 10,000 and 100,000 given in FCA was found nonimmunogenic in rabbits, and PEG of MW 5.9 X 10(6), given s.c. to mice, showed no or very poor immunogenic properties. Gel diffusion, heterologous passive anaphylaxis and passive hemagglutination were used to demonstrate anti-PEG antibodies raised to PEG-modified proteins. Specificity was confirmed by hapten inhibition of precipitation, inhibition of passive hemagglutination and cross-reactivity tests. PEG of MW greater than or equal to 4,000 produced specific precipitates, smaller molecules acted as monovalent haptens. From hapten inhibition of precipitation by PEG of MW 300 it appears that the antigenic determinant of PEG may be a sequence of 6-7 -CH2CH2O-units. Anti-PEG antibodies can be used analytically. By gel diffusion, Peg was detected in minimal concentrations of 0.1-1 microgram/ml. The clinical relevance of these findings with regard to therapy with PEG-modified enzymes and allergens in humans remains to be established.

Animals

Stimulation with a monoclonal antibody (mAb4E4) of scavenger receptor-mediated uptake of chemically modified low density lipoproteins by THP-1-derived macrophages enhances foam cell generation.

mAb4E4, a murine monoclonal antibody that is specific for acetylated LDL and malondialdehyde-treated LDL, binds specifically to modified LDL present in human atherosclerotic lesions. It is directed against an epitope that is poorly exposed in delipidated and solubilized apolipoprotein B-100 from modified LDL. mAb4E4, as well as its F(ab')2 and Fab fragments, enhanced the uptake of both acetylated LDL and malondialdehyde-treated LDL by THP-1-derived macrophages resulting in a sixfold increase of cytoplasmic cholesteryl ester levels. The increased uptake of modified LDL/mAb4E4 complexes did not occur via the Fc receptor and did not depend on aggregation of modified LDL particles. However, their uptake was inhibited by blocking the scavenger receptors with fucoidin or by downregulation of receptor expression with endotoxins or interferon-gamma, indicating that their uptake is mediated via these receptors. Thus, generation of autoimmune antibodies against modified LDL and subsequent endocytosis of soluble modified LDL/antibody complexes via scavenger receptors may enhance foam cell generation. This mechanism may contribute to the progression of atherosclerotic lesions.

1,2-Dipalmitoylphosphatidylcholine

Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease.

The murine monoclonal antibody mAb-1H11 raised against malondialdehyde (MDA)-modified LDL, was used to detect cross-reacting material in human atheromatous tissue and in plasma. MDA-modified LDL levels in plasma were 0.19 +/- 0.02 mg/dl (mean +/- SEM) in 44 control subjects, 0.24 +/- 0.02 mg/dl in 15 patients with chronic stable angina pectoris (P = NS vs LDL cholesterol matched controls), 1.4 +/- 0.1 mg/dl in 60 patients with acute myocardial infarction (P < 0.001 vs controls), and 0.86 +/- 0.11 mg/dl in 22 patients with carotid atherosclerosis (P < 0.001 vs controls). Modified LDL, isolated from pooled LDL of 10 patients, showed a higher electrophoretic mobility on agarose gels, a higher content of thiobarbituric acid reactive substances, and a higher cholesterol/protein ratio than native LDL and had a similar reactivity (antigen/protein ratio) in the assay as the in vitro MDA-modified LDL used for calibration. Its apo B-100 moiety was not fragmented. Uptake of this modified LDL by macrophages resulted in foam cell generation. In conclusion, elevated plasma levels of atherogenic MDA-modified LDL may be a marker for unstable atherosclerotic cardiovascular disease.

Aged

Modified release tizanidine: a review.

The pharmacokinetic and clinical properties of a modified release formulation of the alpha 2-adrenergic agonist tizanidine are reviewed. Therapy with conventional tizanidine tablets is effective in the relief of spasticity, but it has a short half-life, resulting in it having to be administered three or four times daily. The modified release capsules have retard characteristics which allow the drug to be given as a once-daily dosage. Single- and multiple-dose pharmacokinetic studies in healthy volunteers have shown that the relative bioavailability of the modified release formulation is similar to that of conventional tablets and is unaffected by food. Clinical studies have shown that modified release tizanidine improved spasticity and disability in approximately 94% and 79%, respectively, of spastic patients. Adverse effects were observed in about 33% of patients. Mild, transient muscular weakness was the most common effect, but it did not require termination of treatment. Body weight, blood pressure, heart rate and haematological or biochemical measures were not adversely affected. The initial recommended dose of modified release tizanidine is one capsule per day of 6 mg, which may be increased gradually to 24 mg. A daily dose of 12 mg modified release tizanidine was used in 50% of patients studied.

Biological Availability

Microtubules rich in modified alpha-tubulin characterize the tail processes of motile fibroblasts.

The organisation of microtubules rich in post-translationally modified alpha-tubulin has been investigated in a fibroblast cell line (NIH-3T3-T15) that can be reversibly transformed. An immunofluorescence microscopy study of the static non-transformed cells has revealed a central distribution of wavy microtubules showing post-translational modifications. When transformed there is a marked increase in cell motility and the appearance of long thin cytoplasmic 'tails'. These tails have been found to contain conspicuous bundles of post-translationally modified microtubules that run down the length of the processes and terminate close to the plasmalemma. Both detyrosinated and acetylated alpha-tubulin are present as major species in these modified microtubules. Such a pattern of modified microtubules is only occasionally seen in the untransformed NIH-3T3-T15 cells. We have also found them to be present in other transformed fibroblast lines. The presence of bundles of microtubules rich in modified alpha-tubulin in the cell tails is correlated with a marked reduction in the numbers of F-actin stress fibres. The possible role of these modified stable microtubules in cell motility is discussed.

Cell Line

Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro.

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.

RNA, Small Nuclear