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PubMed1991

Correlation of amino acid residues in the M1 and M2 proteins of influenza virus with high yielding properties.

The ability of influenza A viruses to replicate to high titer in the allantoic sac of the chicken embryo has been mapped to the matrix protein gene (RNA 7). Because influenza A/WSN/33 (H1N1) virus grows poorly in this host but contains a matrix protein gene with a sequence similar to sequences from viruses that grow well in eggs, we derived a single gene reassortant containing only the M gene from A/WSN/33 (H1N1) in a background of the other 7 RNA segments from A/Philippines/2/82 (H3N2) (a low yielding virus, hy-). This reassortant replicated 10 times better than the A/WSN parent itself, indicating that the high yielding (hy+) phenotype of the A/WSN/33 M gene may be suppressed by one of the other genes of A/WSN/33. Comparison of M gene sequences between hy+ (including A/WSN/33) and hy- strains allowed us to correlate specific amino acid positions in M1 and M2 proteins with the growth properties of influenza viruses.

Amino Acid Sequence↗
PubMed2004

Hepatitis B virus genotype A and D and clinical outcomes of liver transplantation for HBV-related disease.

Hepatitis B virus (HBV) genotypes have been associated with specific patterns of disease and response to antiviral therapy. We investigated the effect of HBV genotype on HBV recurrence and mortality after liver transplantation (LT). Pretransplant sera of 45 hepatitis B surface antigen (HBsAg) positive adults were submitted for HBV genotyping by a reverse-phase hybridization line probe assay with genotype-specific probes. Data were correlated with clinical outcomes after transplantation. Genotype A (n =15), D (n = 13) and A/D (n = 12) accounted for 89% of all genotypes. Coinfection with two HBV genotypes was encountered in 14 (31.1%) patients. Eighteen patients (40 %) developed HBV recurrence at a median of 10 months posttransplant (range, 1-53) and 10 patients (22 %) died at a median of 24 months (range, 3-63). Genotype D patients were more likely to develop HBV recurrence or die compared with genotype A patients, although this did not reach statistical significance. Dual infection with genotype A/D resulted in mortality similar to that of genotype A but recurrence similar to that of genotype D. Active viral replication at time of transplantation was the only independent factor (P = 0.03) that predicted HBV recurrence. In conclusion, HBV genotype A and D did not have a significant impact on clinical outcomes of LT for HBV-related liver disease in patients of European origin. These data do not support routine HBV genotyping in liver transplantation.

Adolescent↗
PubMed1981

Determinants of susceptibility and resistance to feline leukemia virus infection. II. Susceptibility of feline lymphocytes to productive feline leukemia virus infection.

The interplay between feline leukemia virus (FeLV) and feline lymphocytes (lc) infected in vitro or in vivo was investigated. Surface marker analysis and viral infectivity (VI) assays of lc populations were used to determine susceptibility of lc subsets to FeLV. The principal FeLV-replicating cell in the mesenteric lymph node of persistently infected, preleukemic cats was a nonadherent, complement receptor (CR)-bearing lc (B-cell). The lymph nodes of preleukemic cats also had increased numbers of uninfected T-cells [cells forming rosettes with guinea pig erythrocytes (GPE)] and cells with receptors for the Fc portion of 7S IgG (Fc gamma R cells) as compared with lymph nodes of age-matched specific-pathogen-free (SPF) cats. The induction of productive infection of feline peripheral blood mononuclear leukocytes (PBL) in vitro depended on a 48-hour in vitro preincubation period before virus exposure. The equivalent susceptibility of whole and adherent cell-depleted PBL to productive infection and the failure of hydrocortisone to enhance viral infection were compatible with identification of the FeLV-replicating cell as an lc. Furthermore, lc from susceptible SPF kittens replicated 50 times as much FeLV as did lc from resistant adult SPF cats. The Ic productively infected with FeLV after in vitro exposure were more precisely identified with the use of Ficoll-Isopaque density gradient separations of rosetted and nonrosetted lc. Whole PBL, GPE rosette-positive PBL (T-cells), and CR-positive PBL (B-cells) were permissive to FeLV infection, and maximal VI was evident at 14 days after exposure. The substantial (1,325-fold) increment in VI found in the Fc gamma R-depleted PBL suggested a role for Fc gamma R cells in the containment of FeLV infection. Unstimulated mononuclear leukocytes from blood, spleen, lymph node, thymus, and marrow were susceptible to productive FeLV infection after in vitro exposure. The degree of spontaneous DNA synthesis in marrow, thymus, and spleen but not lymph node or PBL was inversely related to permissiveness to viral replication. Mitogen activation of lc was associated with decreased viral replication when either T-cell mitogens (concanavalin A, phytohemagglutinin, or pokeweed mitogen) or a B-cell mitogen (dextran sulfate) was used. Virus production by spleen cells and PBL was enhanced twofold to tenfold by prior lc stimulation by the B-cell mitogen, lipopolysaccharide, or protein A-bearing Staphylococcus aureus, a mitogen for feline T-cells with Fc gamma R. Both productively infected (preincubated) and nonproductively infected (freshly isolated) PBL transferred infectious FeLV to autochthonous peritoneal macrophages (M theta); most of the virus in PBL-peritoneal M theta cocultures was produced by adherent cells, irrespective of whether the adherent or nonadherent cell population was inoculated originally.

Age Factors↗
PubMed1993

Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence.

The mutagenic potential of O4-methylthymine (m4T) and O4-ethylthymine (e4T) was determined by a primer extension assay on a 25mer oligonucleotide containing a single site-specifically incorporated modified thymine. The e4T-containing oligonucleotide was prepared by using a new synthetic procedure suitable for large alkyl groups on thymine. The second-order rate constants, K(app)m and V(rel)max, permitted calculation of the frequency of formation and extension of modified base pairs compared to Watson-Crick pairing. With both m4T and e4T, the T.G type pairing was formed at least 10-fold more frequently than the nonmutagenic alkyl T.A pairing. However, there was a small but reproducible preference for m4T.G pairing. In both cases T-->C transitions would result. There was no evidence for formation of alkyl T.C or T.T. These data suggest that reported T-->A transversions by ethylation are not likely to result from O4-alkylthymine. In contrast to insertion, extension beyond alkylthymine under kinetic conditions did not occur with alkyl T.A. but only with the alkyl T.G termini. For this latter T.G type pairing, the larger ethyl group did not hinder extension compared to that of the methyl group, in the sequence studied. Under non-limiting conditions of dNTP concentration and time, complete replication could be demonstrated for both methyl- and ethyl-containing oligonucleotides. We conclude that the increase in size of the alkyl group from methyl to ethyl does not significantly affect the mutagenic potential and type of mutations of O4-alkylthymine in vitro.

Adenosine Triphosphate↗
PubMed2005

Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu(2+)-deficient or an active Cu,Zn-superoxide dismutase.

Yeast overexpressing SOD1, the gene for Cu,Zn-superoxide dismutase (Cu,Zn-Sod), was used to determine how Sod1p overexpression influences the chronological lifespan [the survival of non-dividing stationary (G0) phase cells over time], the replicative lifespan (the number of buds produced by actively dividing yeast cells) and stress resistance. Increasing the level of active Cu,Zn-Sod in yeast was found to require either growth in the presence of high copper, or the simultaneous overexpression of both SOD1 and CCS1 (the latter being the gene that encodes the chaperone dedicated to Cu(2+)-loading of Sod1p in vivo). Dual SOD1 + CCS1 overexpression elevated the levels of Cu,Zn-Sod activity six- to eight-fold in vegetative cultures. It also increased the optimized survival of stationary cells up to two-fold, showing this chronological lifespan is ultimately limited by oxidative stress. In contrast, several detrimental effects resulted when the SOD1 gene was overexpressed in the absence of either high copper or a simultaneous overexpression of CCS1. Both the chronological and the replicative lifespans were shortened; the cells displayed an abnormally high level of endogenous oxidative stress, resulting in a high rate of spontaneous mutation. Such harmful effects were all reversed through the overexpression of CCS1. It is apparent therefore that they relate to the incomplete Cu(2+)-loading of the overexpressed Sod1p, most probably accumulation of a Cu(2+)-deficient Sod1p to appreciable levels in vivo. The same events may generate the detrimental effects that are frequently, though not universally, observed when Cu,Zn-Sod overexpression is attempted in metazoans.

Catalase↗
PubMed2003

Elongation of oligonucleotide primers forming a triple helix on double-stranded DNA templates by purified DNA polymerases.

Current knowledge on the replication of DNA involves enzymatic steps of DNA strand separation upon helicase activity, thus enabling the exposed bases of the single-stranded DNA to direct the polymerization of complementary nucleotides through Watson-Crick base pairing rules by DNA-dependent DNA polymerases. Here, we report that oligonucleotide primers (9-11 nt long) bound to the double-stranded DNA, can be elongated by the T7 and by the Thermus thermophilus DNA polymerases and by the Escherichia coli Klenow fragment. A perfectly base-paired DNA cannot be used as a template, but a single A/A mismatch located even 5 bp upstream from the theoretical 3'-end of the oligonucleotide primer is sufficient for DNA elongation by these first two enzymes, while five are required for the Klenow fragment. Elongation products from templates containing A/A mismatches at different positions revealed similar patterns independently of the positions of the A/A mismatches. The sequencing of the elongated products revealed that both purine and pyrimidine are incorporated at the pyrimidine-purine-pyrimidine transitions of the template strands, probably because of a shift of the primers on the double helix template. These data confirm that prokaryotic DNA polymerases may accommodate transiently three DNA strands in their catalytic centre. They also show for the first time that replication can occur on double-stranded DNA in the absence of DNA helicase, when mismatches are present in the vicinity of the triple helix initiation complex.

Base Sequence↗
PubMed1986

The use of syntactic information in filling gaps.

Two sentence-comprehension time experiments replicated and extended previously reported research indicating that readers initially make a quick, heuristic assignment of fillers to gaps in temporarily ambiguous sentences such as "Who did John beg to sing?" and "Who did John beg to sing for?" The Most Recent Filler heuristic readers seemed to adopt made the former "distant filler" sentence harder to comprehend than the latter "recent filler" sentence. Readers did not in general use all available sources of information in making this assignment. In particular, they usually delayed their use of verb control information so that substituting the unambiguous-control verbs "force" or "begin" for "beg" did not eliminate the distant filler inferiority. The experiments counter some criticisms that have been made of the previously reported research. The possibility that readers delay using an interesting natural category of information was raised, and its implications for the mental grammar were considered.

Humans↗
PubMed1998

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein.

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.

Cell Line↗
PubMed1994

Magnetic motor evoked potentials during methohexital anesthesia in the dog.

Magnetic motor evoked potentials (MMEPs) were recorded from the right cranial tibial muscle after magnetic stimulation of the left motor cortex in six dogs sedated with oxymorphone. Anesthesia was induced with an intravenous bolus of 5.5 mg/kg of methohexital and maintained with a methohexital infusion. The dogs inspired 100% oxygen during anesthesia. Blood pressure, heart rate, respiratory rate, esophageal temperature, and end-tidal carbon dioxide tension were recorded. The depth of anesthesia was increased until the amplitude of the MMEP was less than 5% of the control value, and the dogs were then allowed to recover. Every 5 minutes during anesthesia, a blood sample was taken for methohexital assay and at the same time, four replicate MMEPs were recorded. Plasma methohexital levels were significantly (P < 0.05) correlated with heart rate (p = 0.38) and end-tidal carbon dioxide tension (p = 0.49) and negatively correlated with respiratory rate (p = 0.74). There was no significant correlation between blood pressure and methohexital levels. The dogs regained consciousness at a plasma methohexital level of 10.4 +/- 3.8 micrograms/ml (mean +/- SD). The amplitude of the MMEP decreased significantly with increasing methohexital levels. In four dogs, the relationship was reasonably linear. The MMEP disappeared at a plasma methohexital level of 23 +/- 6.6 micrograms/ml. The latency of onset of the MMEP increased significantly from its control value of 14.7 +/- 1.0 ms to 17.5 +/- 1.3 ms at the highest methohexital levels at which MMEPs were recordable. This study demonstrated that MMEPs can be reliably recorded under methohexital anesthesia.

Anesthesia, General↗
PubMed1982

[Electron microscopic study of the in vitro effect of dipyridamol on the pseudorabies virus].

Dipyridamole at a concentration of 50 microM/ml displays no activity on adsorption and penetration of pseudorabies virus in chicken embryonal cells. Furthermore, first stages of virus replication take place within the nucleus, whereas incomplete virus cores defective in DNA content are found within the nucleoplasm at times when the regular viral replication has been finished in controls. Defective pseudorabies virus particles lacking in DNA-content of the core, can be observed at the end of normal replication time. Consequently, the antiviral activity of dipyridamole may be due to blocking of the synthesis or of the incorporation of infectious viral DNA into the virus core.

Animals↗
PubMed2006

NASBA: a novel approach to assess hormonal receptors and ERBB2 status in breast cancer.

In human breast cancer, estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERalpha status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.

Adult↗
PubMed2006

Foot pad dermatitis and hock burn in broiler chickens and degree of inheritance.

A total of 2,118 birds from 2 strains were allocated to 12 groups of 93 to 100 each in 2 time-separated replicates. The development of foot pad dermatitis (FPD) and hock burn (HB) were recorded weekly from d 8 to slaughter on a set sample of live animals (7 per group). In addition, feet and hocks of all birds were investigated at slaughter at either 4, 6 (fast-growing strain), 8, or 10 (slow-growing strain) wk of age. Lesions were scored for both the left and right foot and classified according to a scale from 1 (no lesion) to 9 (very severe lesions) for FPD and from 1 (no lesion) to 3 (very severe lesions) for HB. No FPD lesions and very few low-grade HB lesions were found in chickens from the slow-growing strain. In the fast-growing strain, the first signs of FPD and HB were seen in wk 2. The incidence of both types of lesions increased thereafter. Foot pad dermatitis was more frequent in females (49 vs. 36%, P < 0.05). Body weight did not affect FPD, but more HB were found at higher BW (P < 0.01). Egg weight influenced neither FPD nor HB. Variance and covariance components were analyzed using a multivariate animal model, in which scores for FPD and HB were transformed into logarithmic scale. The analyses were carried out using restricted maximum likelihood algorithm. Heritabilities were estimated to be 0.31 +/- 0.12 (SE) for FPD, 0.08 +/- 0.08 for HB, and 0.38 +/- 0.13 for BW. Genetic correlations among these traits were low and nonsignificant. Phenotypic correlation between BW and FPD was low and nonsignificant and between BW and HB was 0.17 +/- 0.05 (P < 0.01). The relative high heritability of FPD and the low genetic correlation to BW suggested that genetic selection against susceptibility to FPD should be possible without negative effects on BW gain.

Analysis of Variance↗
PubMed2005

Effective gene therapy of biliary tract cancers by a conditionally replicative adenovirus expressing uracil phosphoribosyltransferase: significance of timing of 5-fluorouracil administration.

In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for uracil phosphoribosyltransferase (UPRT), which converts 5-fluorouracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.

Adenocarcinoma↗
PubMed1982

Programming time as a function of response duration: a replication of "dit-dah" without possible guessing artifacts.

Reaction time was longer prior to longer-lasting pressing responses for low probability, unanticipated movements. By contrast, reaction time was independent of response duration for high probability, anticipated movements. This replicates and extends previous findings for choice and simple reaction time in the "dit-dah" paradigm. The present results (unanticipated responses) cannot be given a selective anticipation interpretation because the comparison is between responses which are never anticipated. The previous paradigm was open to an alternative interpretation in which selective anticipation of dit rather than dah could have produced the differences in choice reaction time. Confirmation of the finding that variations in required response duration lead to differences in reaction time supports the view that the underlying parameter of "complexity" in the "memory drum theory" may be response duration.

Journal Article↗
PubMed1983

Cell division in Escherichia coli after changes in the velocity of DNA replication.

A method of computer analysis was developed to evaluate the kinetic changes in the rate of cell division in non-synchronous cultures of E. coli resulting from changes in the velocity or initiation of chromosome replication. This method takes into account that the cell division pathway in E. coli includes a reaction of indeterminate length described by a probability function that applies to the cell population. The analysis yields a hypothetical cell number kinetics as it would be observed if the stochastic element in the division pathway were absent. Since this derived cell number curve responds to experimentally induced perturbations of replication at defined times whereas the actual cell number curve reflects these perturbations only in a blurred fashion, replication and division events can be precisely correlated with this method. The method was applied to the evaluation of thymine starvation experiments with two Thy- derivatives of E. coli B/r; one of the strains has a mutationally altered (60% increased) cell mass at initiation of chromosome replication. In both strains, the stochastic phase of the cell cycle had the same half-life value of 10 min and began 18 min after each termination of replication. This suggests that the time of cell division is linked to replication, not to cell mass or length. This interpretation is supported by results of experiments in which the rate of cell growth was altered at the time of thymine starvation.

Cell Division↗
PubMed2000

Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.

The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.

Cell Nucleus↗
PubMed1993

Chromosome bands--flavours to savour.

The mammalian chromosome is longitudinally heterogeneous in structure and function and this is the basis for the specific banding patterns produced by various chromosome staining techniques. The two most frequently used techniques are G, or Giemsa banding and R, or reverse banding. Each type of stained band is characterised by variations in gene density, time of replication, base composition, density of repeat sequences, and chromatin packaging. It is increasingly apparent that R and G bands, which are complementary to each other, represent separate compartments of the euchromatic human genome, with R bands containing the vast majority of genes. R bands are also more GC-rich, contain a higher density of Alu repeats, and replicate earlier in S phase, than G bands. These properties may be interdependent and may have coevolved.

Base Composition↗