Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

The dilemma of negative analysis of variance estimators of intraclass correlation

Summary At least two common practices exist when a negative variance component estimate is obtained, either setting it to zero or not reporting the estimate. The consequences of these practices are investigated in the context of the intraclass correlation estimation in terms of bias, variance and mean squared error (MSE). For the one-way analysis of variance random effects model and its extension to the common correlation model, we compare five estimators: analysis of variance (ANOVA), concentrated ANOVA, truncated ANOVA and two maximum likelihood-like (ML) estimators. For the balanced case, the exact bias and MSE are calculated via numerical integration of the exact sample distributions, while a Monte Carlo simulation study is conducted for the unbalanced case. The results indicate that the ANOVA estimator performs well except for designs with family size n = 2. The two ML estimators are generally poor, and the concentrated and truncated ANOVA estimators have some advantages over the ANOVA in terms of MSE. However, the large biases may make the concentrated and truncated ANOVA estimators objectionable when intraclass correlation () is small. Bias should be a concern when a pooled estimate is obtained from the literature since <0.05 in many genetic studies.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (<Emphasis Type="Italic">Helianthus annum</Emphasis>, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented.Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.