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PubMed1990

Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA.

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1976

Cation control of chlorophyll a fluorescence yield in chloroplasts. Location of cation sensitive sites.

We have compared the effect of various cations on the fluorescence yield of chloroplasts under several different conditions in vitro. 1. In the absence of a high energy state but in the presence of low (10 mM) concentrations of monovalent cations, divalent metal cations increase steady state chlorophyll a fluorescence yield in a manner which does not involve transport of these cations across the thylakoid membranes. 2. The kinetics of this cation-induced fluorescence rise are relatively slow, and seem to reflect cation binding (or subsequent conformational changes) to sites on the outer surface of the thylakoid membrane. 3. In the absence of monovalent cations, the apparent binding constant for Mg2+ to sites on the outer side of the membrane is low. Addition of low concentrations of monovalent cations (10 mM) competitively inhibits divalent cation binding. 4. Control of fluorescence yield is also exerted by the high energy state, and seems to involve proton/metal cation exchange at sites on the inner side of the thylakoid. 5. When isolated chloroplasts are washed and resuspended in a medium containing no added cations, the initial fluorescence level is high, but is quenched on addition of monovalent cations, sodium EDTA being much more effective than sodium chloride. It is argued that when isolated under these conditions, chloroplasts retain sufficient divalent cations to saturate external negative sites, and that the fluorescence lowering is due to their removal. 6. Some other cations, such as poly(L-lysine), can displace divalent cations from their external sites in an irreversible manner, resulting in a fixed low fluorescence yield.

Binding, Competitive↗
PubMed1976

Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 mus range in Chlorella pyyrenoidosa during incubation with hydroxylamine.

Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0-16 mus were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 mus following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time td, the steady-state oxygen yield in the flashes is exp(-ktd), the yield at td=0 being taken equal to 1, where k=(0.1 + beta[NH2OH])s-1, with [NH2OH] in mM and beta=0.6 mM-1, provided [NH2OH]greater than or equal to 0.5 mM. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P+ occurs, proceeding as exp (-kiti)where ti is the incubation time with hydroxylamine and ki=(alpha[NH2OH]) min-1, with [NH2OH] in mM and alpha=0.14 mM-1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference deltaphi=phi(16 mus)-phi(2 mus) induced by a flesh in the steady state. In a fraction of the reaction centers this inhibition occurs "immediately" after the addition of hydroxylamine. These observations, combined with the conslusion of Cheniae and Martin (1971, Plant Physiol. 47, 568-575) that the inhibition of the Hill reaction is related to the extraction of bound manganese indicate that the reaction between Z and P+ requires bound manganese. 3. In the inhibited centers a second donor for P+, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample.

Chlorella↗
PubMed1979

Comparative study of the fluorescence yield and of the C550 absorption change at room temperature.

The C550 absorption change and the fluorescence yield were studied at room temperature in chloroplasts in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and under conditions in which contributions of P-700 and of the electrochromic effect were neglible. 1. The C550 difference spectrum is a typical band shift with an isobestic point close to 550 nm. 2. The maximum amplitude of C550 absorption change is reached upon the first flash of a series of saturating flashes, unlike the maximum fluorescence yield which is attained after several flashes. 3. The comparison of the induction curves of the C550 change and the fluorescence yield in weak light shows that the fluorescence yield is controlled by two quenchers: one of them (Q1), the redox state of which C550 is a probe, is responsible for the major part of the quenching; the other one (Q2), which is less concentrated and less efficient becomes predominant at the end of the fluorescence induction. 4. Quencher Q2 back-reacts faster than quencher Q1. 5. Two alternative models are discussed in which Q1 and Q2 belong either to the same Photosystem II center or to two different photocenters.

Chloroplasts↗
PubMed1990

Low concentrations of macromolecular solutes significantly affect electrofusion yield in erythrocyte ghosts.

Electrofusion yields in rabbit erythrocyte ghosts containing various amounts of hemoglobin, bovine serum albumin, or dextran at low concentrations were measured as a function of pulse field strength and pulse decay half-time. The presence of any of the macromolecules in low concentrations caused fusion yields to be significantly higher than when the ghosts were white (i.e., containing only buffer). The fusion yield enhancement was also critically dependent on the parameters of the electric field pulse. The fusion yield was also significantly affected by small changes in the concentration of hemoglobin when it was present outside the ghost membranes in the suspension buffer.

Animals↗
PubMed1983

A high yield method for the preparative synthesis of coenzyme A by combination of chemical and enzymic reactions.

Dried cells of Brevibacterium ammoniagenes are a good enzyme source for the preparative synthesis of CoA from pantothenic acid, L-cysteine and ATP. A problem with this synthesis is that the CoA synthesis is repressed by negative feedback inhibition by CoA to pantothenate kinase, the first step enzyme for the biosynthesis of CoA, which catalyses phosphorylation of pantothenic acid or pantetheine. As the inhibition operates only at this step, a further increased yield is possible if the enzymic phosphorylation step is replaced with chemical synthesis. Yields from phosphorylated substrates are more than 10-times higher than those from pantothenic acid or pantetheine (33 g/l from phosphopantothenic acid with a molar yield of 86%; 115 g/l from phosphopantetheine with a molar yield of 100%).

Brevibacterium↗
PubMed1986

Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis.

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

Ammonium Sulfate↗
PubMed1983

Mutagenesis and anti-mutagenesis in Salmonella: influence of ethionine and caffeine on yields of mutations induced by 2-aminopurine and 9-aminoacridine.

Ethionine, the ethyl analogue of methionine, slightly reduced the yield of reversions of the hisC3076 frameshift marker induced by 9-aminoacridine (9AA) in an excision-proficient strain of Salmonella typhimurium, but completely abolished mutagenesis by 9AA in the excision-deficient uvrB-deletion strain TA1537. No toxic effects of ethionine were apparent in either the excision-proficient or the excision-deficient strain. Because of the differential effects of ethionine on mutagenesis in the two strains, it seemed possible that an ethionine-sensitive step in the process(es) leading to fixation of 9AA-induced mutations might be compensated for by the uvrA,B,C+ excision-repair system. To further test this possibility, we used caffeine (a compound known to significantly reduce the efficacy of the excision-repair process) as a co-treatment with ethionine for cells of an excision-proficient strain exposed to 9AA. Treatment with caffeine alone or ethionine alone had very little effect on reversion yield, whereas co-treatment with the two agents abolished 9AA mutagenesis. It appeared, therefore, that either the caffeine-sensitive pathway or the ethionine-sensitive pathway needed to be functioning if 9AA-induced reversions of hisC3076 marker were to be detected. Addition of methionine to cells of the excision-deficient strain exposed to 9AA restored their ability to be mutated by 9AA, however. In a base-pair substitution back-mutation system, ethionine slightly enhanced the yields of revertants of the trpE8 marker induced by 2-aminopurine (2AP) in both an excision-proficient strain (at all 2AP dose levels tested) and an excision-deficient strain (only at the lower dose levels). In the excision-deficient strain, doses of 2AP above 300 micrograms/plate were highly toxic when ethionine was also present. It was for this reason that no 2AP-induced revertants were recovered at the higher 2AP concentrations. Treatment of the trpE8 strain with methionine also enhanced the yield of 2AP-induced revertants of this marker.

2-Aminopurine↗
PubMed1986

A critical comparison of the micronucleus yield from high and low LET irradiation of plateau-phase cell populations.

Experimental data are presented which cast doubt on the usefulness of micronucleus assays as a quantitative measure of radiation damage. Synchronised (G1) Syrian hamster fibroblasts (BHK21 C13) were exposed to doses of gamma- or neutron radiation which yielded equivalent survival response. The cultures were examined at intervals during a 120-h post-irradiation incubation, for the appearance of micronuclei. Dose-response curves for the micronucleus yield constructed at a single sample time of 30 h were compared with those for the peak yield, irrespective of sampling time at each dose. When the total production of acentric fragments was compared with the peak yield of micronuclei no clear correlation could be seen. Qualitative hypotheses have been suggested to account for the various features of the data. The production and expression of micronuclei has been found to be a very complicated relationship and is a warning against too simplistic an interpretation of micronuclei data.

Animals↗
PubMed1992

Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells.

Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 30 h after treatment for 3 h with or without S9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine beta-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 microM, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 microM ADR were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.

Aminobiphenyl Compounds↗
PubMed1985

ESS germination strategies in randomly varying environments. II. Reciprocal Yield-Law models.

The study of ESS germination fractions in S. Ellner (1985, Theor. Pop. Biol. 27, 000-000) is applied and extended in the case of annuals obeying the Reciprocal Yield Law. The effects of parameter changes on the value of the ESS germination fraction are determined in some limiting cases by analyses of approximations. Numerical solutions of the ESS criterion are used to check the robustness of the conclusions and the accuracy of the approximations. In general, the ESS germination fraction decreases with an increased survivorship of buried seeds and with increased "variability" of seed yields. However, different measures of "variability" are appropriate in different circumstances. To avoid the possibility of conflicting predictions depending on the measure of variability, it is suggested that tests of the theory be limited to co-occurring species, and to variability due to climatic fluctuations. The ESS theory based on the Reciprocal Yield Law is compared with D. Cohen's (1966, J. Theor. Biol. 12, 119-129; 1968, J. Ecol. 56, 219-228) density-independent theory of "optimal" germination. The theories differ qualitatively and quantitatively regarding the influence of mean yield, seed survivorship, and the frequency of favorable years on the predicted germination fraction.

Biological Evolution↗
PubMed1989

Identification of PR8 M1 protein in influenza virus high-yield reassortants by M1-specific monoclonal antibodies.

A panel of monoclonal antibodies to the M1 protein of A/PR8/34 (H1N1) (PR8) influenza A virus was found to distinguish in ELISA high-yielding reassortant viruses derived from reassortment of PR8 and X-31 (H3N2) viruses with recently prevalent field strains of H1N1 or H3N2 subtype. These findings are concordant with results of genotyping that demonstrated the presence of PR8 RNA 7 or M1 protein in high-yield reassortants by RNA or protein PAGE. All high-yield vaccine candidate reassortants Application of the M1 monoclonal antibody panel facilitates the isolation of high-yield vaccine candidate reassortants bearing the PR8 M1 gene, and should aid in epidemiologic strain tracking as well.

Antibodies, Monoclonal↗
PubMed1992

Predictive relationships for sidestream smoke cigarette yields.

Relationships have been sought for smoking machine generated sidestream smoke yields of cigarettes for particulate matter (water and nicotine free) (PMWNF) and nicotine. Comparisons have been made with the corresponding mainstream smoke yields, other readily measurable factors in machine smoking and with the physical characteristics of cigarettes. The sidestream smoke yields have been found to be related to the puff count during machine smoking multiplied by the cross-sectional area of a cigarette or to the total weight of cigarette tobacco burnt during machine smoking. For the brands retailed in the U.K. these relationships permit calculation of the sidestream smoke contributions of PMWNF and nicotine to environmental tobacco smoke. From the data used to derive these relationships, and based on 31 cigarette brands representing 71% of cigarette sales in the U.K., the sales weighted sidestream PMWNF yield is 27.1 mg/cigarette and that for nicotine is 5.12 mg/cigarette.

Humans↗
PubMed1989

Effect of harvest medium composition on yield and chain nature of recombinant tissue-type plasminogen activator species.

The effect of harvest medium composition on yield and quality of two recombinant tissue type plasminogen activators (t-PA) has been examined. Protein hydrolysates increased the yield of native t-PA up to 3-fold compared to control cultures. In marked contrast, the yield of a growth-factor deleted t-PA mutant was adversely affected by the use of these additives. Partially purified t-PA that had been harvested in the presence of protein hydrolysates had higher fibrin plate activity: amidolytic activity ratios. In addition Western blotting showed these preparations contained less degraded material. These observations suggest that protein hydrolysates have opposite effects on two closely related recombinant cell lines and that they can affect quality as well as yield of secreted proteins.

Animals↗
PubMed1990

Population characteristics and cigarette yield as determinants of smoke exposure.

Relationships of population characteristics, smoking history, and cigarette yield with smoke exposure as measured by peripheral blood concentrations of thiocyanate, carboxyhemoglobin, nicotine and cotinine were sought in 170 male smokers. This population of smokers had significant elevations of serum thiocyanate, blood carboxyhemoglobin and plasma nicotine and cotinine concentrations as compared with an equal number of age- and sex-matched nonsmokers and these concentrations correlated significantly with past 24-hour cigarette consumption. Although the nicotine yield of the cigarette correlated significantly with plasma cotinine and marginally with plasma nicotine, the reduction in plasma nicotine and cotinine was not proportionate to the reduced yield of the cigarettes, suggesting that smokers partially compensate for the lower yields of their cigarettes. Blood levels of carboxyhemoglobin, nicotine and cotinine were also significantly associated with the weight of the subjects, presumably due to the relationship between weight and the volume of distribution. Univariate and multiple regression analyses provided evidence that coffee and alcohol consumption and years smoked also may be important determinants of smoke exposure.

Adult↗
PubMed1989

Lung cancer risk is proportional to cigarette tar yield: evidence from a prospective study.

The age-adjusted risk for lung cancer among over 120,000 male current cigarette smokers in the American Cancer Society's 1959-1972 prospective study was analyzed according to tar yield and quantity smoked per day. At each quantity level, the risk increased with increasing tar yield, and at each tar level, the risk increased with numbers of cigarettes smoked daily. The risks in smokers of cigarettes with the lowest yields, however, far exceeded those of former smokers and nonsmokers. The excess lung cancer risk for current smokers was directly proportional to the estimated total milligrams of tar consumed daily: SMR = 100 + 1.731 x milligrams tar per day. Tar yields today are much lower than they were at the time of this study and presage an eventual reduction (but not elimination) of lung cancer risk for those who continue to smoke cigarettes, especially among lifetime smokers of low-tar cigarettes.

Humans↗
PubMed1991

Pattern of sex steroids secretion and their relationship with embryo yield in Jersey cows superovulated with PMSG.

The levels of progesterone and estrogen secretion were studied in relationship to the superovulatory response in Jersey cows. Progesterone and estrogen concentrations were measured in superovulated Jersey cows with the objective of correlating the patterns of steroid secretion with embryo yield and quality. Pregnant mare serum gonadotropin (PMSG) was used in combination with prostaglandin F(2) alpha analogue to induce superovulation in 18 multiparous, cyclic cows. Serum progesterone and estradiol levels from cows which exhibited estrus within 24 to 48 h after prostaglandin administration (n=13) were used to estimate the superovulatory response. Sex steroid concentrations at the day of estrus (Day 0) was a strong indicator of embryo yield. Progesterone was negatively (r=-0.56) and estrogen positively (r=0.80) correlated to the number of embryos collected. Dramatic increase in progesterone from Day 0 to Day 7 was a significant indicator of embryo yield. A higher rise of estrogen in the follicular phase was an indicator of a larger number of growing follicles and, consequently, better superovulatory response. Nonresponding animals did not show any significant change in the hormonal profile from the day of PMSG treatment to the day of embryo collection. The estimation of progesterone and estradiol concentrations, simultaneously, gave a more objective prediction of embryo yield.

Journal Article↗
PubMed1993

Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system.

A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system. Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth. Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate. In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF). The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI). At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection. The yield of rhNGF was 20 mg l-1. The fed-batch method was scaled up to 12 l stirred bioreactor.

Animals↗