Biomedical subjects

A P Wagner

Publications and source records attributed to A P Wagner.

At least 19 recordsLinked to original sources

PubMed2006

Estimating relatedness and relationships using microsatellite loci with null alleles.

Relatedness is often estimated from microsatellite genotypes that include null alleles. When null alleles are present, observed genotypes represent one of several possible true genotypes. If null alleles are detected, but analyses do not adjust for their presence (ie, observed genotypes are treated as true genotypes), then estimates of relatedness and relationship can be incorrect. The number of loci available in many wildlife studies is limited, and loci with null alleles are commonly a large proportion of data that cannot be discarded without substantial loss of power. To resolve this problem, we present a new approach for estimating relatedness and relationships from data sets that include null alleles. Once it is recognized that the probability of the observed genotypes is dependent on the probabilities of a limited number of possible true genotypes, the required adjustments are straightforward. The concept can be applied to any existing estimators of relatedness and relationships. We review established maximum likelihood estimators and apply the correction in that setting. In an application of the corrected method to data from striped hyenas, we demonstrate that correcting for the presence of null alleles affect results substantially. Finally, we use simulated data to confirm that this method works better than two common approaches, namely ignoring the presence of null alleles or discarding affected loci.

Alleles↗

PubMed2001

Dynamics of gene expression for immediate early- and late genes after seizure activity in aged rats.

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator ( tPA), is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure, and the levels of c-fos and tPA mRNAs induction are decreased in the cortex and in the hippocampus of 30 month old rats, as compared to the levels expressed by 3 month old rats. In addition, at the peak induction, the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes, we found that there was a shift toward earlier times in the maximum expression of growth-related molecules, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18 month old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (1) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (2) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Journal Article↗

PubMed2000

Brain plasticity: to what extent do aged animals retain the capacity to coordinate gene activity in response to acute challenges.

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator (TPA) is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure and the levels of c-fos and TPA mRNAs induction are decreased in the cortex and in the hippocampus of 30-month-old rats, as compared to the levels expressed by 3-month-old rats. In addition, at the peak induction the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes we found that there was a shift toward earlier times in the maximum expression of growth-related molecule, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18-month-old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (i) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (ii) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Aging↗

PubMed1998

Altered expression of microtubule-associated protein 1B in cerebral cortical structures of pentylenetetrazole-treated rats.

Using Northern blot, immunoblotting, immunocytochemistry, and in situ hybridization, we show that a single administration of the convulsant pentylenetetrazole leads to robust, long-term changes in microtubule-associated protein 1B and its mRNA, in the adult rat brain. The first increases in MAP1B mRNA were detected at 15 hr following pentylenetetrazole administration in the temporal (Te2) and perirhinal cortex followed by increases in microtubule-associated protein 1B immunoreactivity at 72 hr postseizure. In contrast, the levels of microtubule-associated protein 1B mRNA and protein in layers I-II of the retrosplenial and parietal cortex (Par2) declined visibly by 24 hr and 72 h, respectively, post-seizure. The changes included loss of staining in layers I-II and development of structures resembling "strings-of-beads" along the fibers of projection neurons of layer V. The levels of microtubule-associated protein 1B mRNA in the entorhinal cortex peaked at later times (72 h), especially in layers II-III, and returned to control levels by 10 days. Whereas the levels of microtubule-associated protein 1B immunoreactivity in the retrosplenial and parietal cortex recovered by 5-10 days, it persisted at high levels through day 35 in layer V of the temporal cortex (Te2), layers II-III of the perirhinal cortex and layers I-II of the lateral entorhinal cortex. These results indicate that seizure activity leads to long-term upregulation of genes coding for structural elements that are characteristic of the immature brain such as microtubule-associated protein 1B.

Animals↗

PubMed1997

Needle placement with transtympanic electrocochleography.

Electrocochleography (ECoG) is an objective, electrophysiologic test useful in the clinical diagnosis of endolymphatic hydrops, or Meniere's disease. The purpose of this study was to determine if the position of the needle, using transtympanic methodology, gives a variable SP/AP (summating potential/action potential) response. SP/AP ratios were obtained during routine tympanoplasty procedures. After the tympanic membrane remnant was removed using a lateral graft technique, precise needle placement was obtained at the medial and lateral round window niches, as well as on the promontory. SP/AP ratios were obtained in these three needle positions. There was no significant difference in the SP/AP ratio responses despite the location of needle placement. The use of transtympanic electrocochleography can give very good wave form morphology and consistent results. Therefore, if elevated SP/AP ratios do occur, they are thought to be due to a pathologic process of the ear and not needle placement.

Audiometry, Evoked Response↗

PubMed1992

Dynamics of gene expression for microtubule-associated protein MAP1B, embryonic alpha-tubulin and late neural beta-tubulin mRNAs in the hippocampus of aged rats.

In the present study we characterized the developmental changes in the prevalence of mRNA coding for microtubule-associated protein, MAP1B, embryonic alpha-tubulin and late neural beta-tubulin in rat hippocampus and forebrain from 1 to 720 days of age using RNA gel-blot analysis. We find that (i) the microtubule-associated protein, MAP1B, signal was relatively abundant at early postnatal stages when compared with mature animals. The hybridization signal in the 24-month-old rats was was approximately 1.7 times that observed in 6-month-old rats. (ii) Embryonic alpha-tubulin and late neural beta-tubulin were differentially regulated during rat brain development. This regulation is characterized by a dramatic decrease in the amount of alpha-tubulin after day-1 and a coincident increase in the production of late neural beta-tubulin. Both messages became stabilized at moderate levels during the subsequent developmental stages. However, the averaged signal for beta-tubulin was then approximately 1.8-fold increased in 24- vs. 6-month-old rats. These results are consistent with hypothesis of an age-associated increase in reactive synaptogenesis where the healthy neurons sprout new connections to compensate for neuronal loss occurring in neighboring neurons.

Aging↗

PubMed1992

Neural cell adhesion molecule (NCAM) and N-cadherin mRNA during development and aging: selective reduction in the 7.4-kb and 6.7-kb NCAM mRNA levels in the hippocampus of adult and old rats.

By examining the time course, from E15 to 720 days of age, for changes in the prevalence of mRNAs coding for neural cell adhesion molecule (NCAM), N-cadherin and alpha-tubulin in rat hippocampus and forebrain, it was concluded that (i) the NCAM 7.4-, 6.7-, 5.2-, 4.3- and 2.9-kb mRNAs are differentially regulated during development and aging; (ii) the 7.4- and 6.7-kb mRNA are drastically reduced starting from day 21 onward; (iii) the E15- and day-1-specific mRNA of 4.3 kb is replaced with the 5.2-kb mRNA starting with 21 days, thereafter the 5.2-kb message remained relatively constant over the entire life-span studied. Likewise, the 2.9-kb mRNA, which was very abundantly expressed at E15 and early postnatal stages, remained relatively constant between 180 days and 720 days; (iv) postnatal rat brains showed both qualitative and quantitative changes in N-cadherin 4.3- and 4.0-kb transcripts. The 4.3-kb mRNA was relatively abundant at 1 and 21 days postnatal stages, thereafter the signal remained very low over the entire life-span studied. The 4.0-kb message, which was specific for the E15 stage, was replaced with the 4.3-kb message; (v) as expected, the 1.8-kb mRNA coding for embryonic alpha-tubulin decreased dramatically after 1 day, but became stabilized at moderate levels during the subsequent developmental stages. At least for the NCAM gene, the regulation seems to occur post-transcriptionally, possibly at the level of RNA processing while the N-cadherin mRNA expression seems to be transcriptionally regulated.

Aging↗

PubMed1991

Synthesis of myristoyl-carba(dethia)-coenzyme A and S-(3-oxohexadecyl)-coenzyme A, two potent inhibitors of myristoyl-CoA:protein N-myristoyltransferase.

1. Two non-hydrolysable analogues of myristoyl-coenzyme A were synthesised and spectroscopically characterized. Myristoyl-carba(dethia)coenzyme A was prepared in a multistep synthesis starting from tridecyl vinyl ketone. S-(3-Oxohexadecyl)-coenzyme A was synthesised from 3-oxohexadecyl chloride by direct condensation with coenzyme A. 2. Both analogues were strong competitive inhibitors of N-myristoyltransferase from yeast. Ki values of 0.3 and 0.25 microM were determined for myristoyl-carba(dethia)-coenzyme A and S-(3-oxohexadecyl)-coenzyme A, respectively.

Acyl Coenzyme A↗

PubMed1991

Evidence that the V+ fibronectin mRNA is increased in the hippocampus of aged rats.

In this study we investigated the splicing pattern of fibronectin (FN) mRNA during development and aging by using Northern blot hybridization with probes that either recognize all forms of FN mRNA or that are specific for the different spliced forms (FN-EIIIA, FN-EIIIB, and FN-V). We find that (1) the FNmRNA in the hippocampus of some but not all old rats showed a pattern of splicing similar to that found in the forebrain of 21 day old rats and (2) the hybridization signal for the alternatively spliced FN mRNA containing the EIIIA, EIIIB, and V95 segments was relatively abundant at early postnatal stages but very few transcripts were detected in adult and old rats. However, the hybridization signal for the juvenile FN-V mRNA was markedly increased in some but not all two-year-old rats suggesting that aging is an individual process.

Aging↗

PubMed1990

Assay of N-myristoyl transferase by selective adsorption of myristoyl-coenzyme A on acidic alumina.

We have developed a simple and rapid method for detecting the enzyme myristoyl-CoA:protein N-myristoyl transferase. The enzyme catalyzes the transfer of the myristoyl moiety of myristoyl-CoA to the amino-terminal glycine residue of a peptide (protein). Incorporation of the [14C]myristate into the peptide is quantified after separation of the [14C]myristoyl-peptide from unreacted [14C]myristoyl-CoA by selective adsorption of [14C]myristoyl-CoA on acidic alumina. Optimal assay concentrations were 200 microM synthetic peptide, 1 microM [14C]myristoyl-CoA, 10 mM Tris-HCl/1 mM dithiothreitol/0.1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid/aprotinin (10 micrograms/ml) buffer, pH 7.4, and 1-10 micrograms protein.

Acyl Coenzyme A↗

PubMed1990

On the strategy of directed assembly and its relevance to ageing.

The strategy which Nature has chosen to generate complex, asymmetric structures has been to build on previously established structures and to render the internal chemistry asymmetric by a close association with asymmetrically distributed, stable topological organizers. This is what we call the strategy of directed assembly. Ageing and the strategy of directed assembly seem to be general for biological systems displaying history-dependent development. We believe that it is this strategy which imposes severe limitations on topological organizer turnover rates and, therefore plays a major role in initiating the ageing process.

Aging↗

PubMed1989

On the relationships between the rate of cytoskeletal stable assemblies turnover, stability of the differentiated state, development and aging.

There is a general consensus that biological specificity is a structure-derived property. If a living system is going to maintain its structure and function then the newly synthesized molecules should replace the faulty ones at the correct time and in the correct places so that the previously established cellular topology will be preserved. In addition, pre-existing spatial determinants which will direct the asymmetrical assembly of the newly synthesized molecules should be available. Therefore, regulation of turnover of cellular architecture represents an essential feature of living systems. In considering the underlying causes of cellular senescence it seemed reasonable to focus on the relationship between development of a stable phenotype and the turnover of cellular and extracellular stable assemblies, currently thought to be involved in maintaining the stability of the differentiated state. In recent years evidence has accumulated suggesting a reciprocal relationship between cytoarchitecture turnover rate and achievement of a stable structure. The lack of a feedback control on the turnover of cellular stable assemblies and/or a low turnover rate of cytoarchitecture components would mean that they will be subjected to damaging processes such as oxidation, cross-linking, aminoacid racemization or non-enzymatic browning which are known to occur in other long-lived proteins. The consequence would be the generation, with advancing age, of faulty cellular structures which, in turn, would alter the deposition of newly synthesized molecules. This process may lead to a progressive breakdown in cellular and extracellular stable structures. The process of directed assembly seems to be general for biological systems displaying history-dependent development. We believe that it is this strategy which imposes severe limitations on presegregated spatial determinants turnover rates and, therefore plays a major role in initiating the aging process. We also suggest that species-specific life-span might be determined by the species-specific regulatory networks which governs the cell-specific cytoarchitecture damaging rate. Moreover, aging appears to be an intrinsic feature of biological systems displaying history-dependent development and should be absent in systems displaying history-independent life-cycles, such as bacteria, some species of protozoa, and certain transformed cell lines. An important feature of protein turnover is that this process requires metabolic energy. Therefore, we can expect that structure preservation strategy is a part of a more general energy-saving strategy, a view previously expressed by T.B.L. Kirkwood (Nature, Lond., 1977, 270, 301-304).

Aging↗

PubMed1984

Electrophoretic concentration of proteins in a nonlinear pH gradient.

A method for electrophoretic concentration of differently charged proteins is described. A nonlinear pH gradient is generated by imposing a potential gradient on an electrolyte system composed of (+)H3PO4-valine (pI 6.0)-Servalyte (pH 9-11)-triethylamine(-). Proteins contained in the valine solution accumulate at the interphase formed between the valine solution and the Servalyte solution. This interphase acts as a barrier or liquid membrane to all proteins having isoelectric points in the range 6-9. For proteins having isoelectric points in the range 5-7 valine is replaced by histidine (pI 7.64) and the Servalyte by Pharmalyte, pH 2.5-5.0. Ribonuclease, hexokinase, bovine serum albumin, and hemoglobin were concentrated and recovered from the top of the column using a peristaltic pump. The duration of concentration process was 1-4 h, the length of the run depending on the experiment scale (20 or 100 ml protein solution), the amount of protein, and the isoelectric point of the protein. Proteins were concentrated 9- to 48-fold, depending on the initial volume and concentration of the protein. The recoveries ranged from 79.7 +/- 1.1 for hemoglobin to 93.17 +/- 2.84 for ribonuclease.

Hemoglobins↗

PubMed1983

Single-step, rapid separation of acidic and basic isoenzymes from commercial horseradish peroxidase.

A simple and rapid technique for simultaneous separation of the acidic and basic isoenzymes of horseradish peroxidase is described. Upon application of a potential gradient on the electrolyte system composed of Pharmalyte (pH 2.5-5.0) histidine (pI 7.64), and Ampholyte (pH 9-11), the acidic and basic isoenzymes with pI 4.0-8.4 of horseradish peroxidase accumulated at the two interphases generated by this arrangement, with a recovery of 75 +/- 5%. Some advantages, such as rapidity, simplicity, low cost, good yields, and others, of this system over existing ones are outlined.

Chemical Phenomena↗

PubMed1983

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids.

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNA-Pyronine Y complex which has a different absorption spectrum from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 micrograms/ml Pyronine Y, 0.56 mg/ml RNA, 80 mumol/ml Tris-HCl buffer, pH 7.8, and 2-45 ng/ml RNAase. The effect of complex concentration, pH, molarity and temperature upon the rate of the reaction were determined. The assay is applicable to crude cell-free extracts.

Animals↗

PubMed1983

Age changes of isoelectric points of the molecular forms of tyrosine aminotransferase from rat liver.

Hydrocortisone-induced tyrosine aminotransferase was isolated from liver of young and old rats and purified by ammonium sulfate precipitation, heat treatment, DEAE-cellulose chromatography and isoelectric focusing. Isoelectric points of the molecular forms of the enzyme were as follows: pH 4.30, 5.00, 5.72 for young rats and pH 4.25, 4.7, 6.62 and 8.00 for old rats. At 6 days after the initial homogenization only one major peak in tyrosine aminotransferase activity corresponding to an isoelectric point of pH 5.10 can be recognized.

Aging↗

PubMed1982

Age changes in the H1 group of histones from rat liver.

Histones were obtained from young and old rat livers by extracting them in 0.25 N HCl. They were fractionated on 15% acid urea polyacrylamide gels containing 6.25 M urea and the changes in the ratio of the major histone fractions as a function of age were calculated. Data presented show a significant increase in the amount of H1 degree fraction in the liver of old rats as compared to young rats. This data is discussed and the possible involvement of H1 degree fraction in an increased resistance of old rat liver chromatin to micrococcal nuclease digestion of linker DNA is suggested. Finally, in connection with this increased resistance, some possible consequences in chromatin structure are discussed.

Aging↗

PubMed1982

Age changes of the isoelectric points of non-histone chromosomal proteins from rat liver in the pH range 5 to 8.

Non-histone chromosomal proteins were extracted from purified nuclei of young (2-3 months) and old (24-28 months) rat liver by a two-phase partition method (chloroform:-isoamyl alcohol, 24:1) after histone depletion with 0.25N HCl. The proteins were subjected to analytical isoelectric focusing in the pH range 5-8. On the densitograms derived from Coomasie brilliant blue R-250 stained gels, there were formally four areas separated having the following pH ranges: I) 5.80-6.10; II) 6.25-6.42; III) 6.80-7.00; IV) 7.20-7.35 for young rats, and I) absent; II) 6.35-6.75; III) 6.80-7.00; IV) 7.12-7.40 for old rats. Hence, the main result is the absence of a group of proteins from old rats having the pH range 5.80-6.10. The factors involved in protein separation by isoelectric focusing, like time-dependent post-translational charge modifications: phosphorylation, acetylation, ADP-ribosylation, deamidation, terminal peptide cleavage, glycosylation, and DNA-like contaminants are discussed.

Aging↗