Analysis of autophosphorylating kinase activities of Arabidopsis and human cryptochromes

Cryptochromes are FAD-based blue-light photoreceptors that regulate growth and development in plants and the circadian clock in animals. Arabidopsis thaliana and humans possess two cryptochromes. Recently, it was found that Arabidopsis cryptochrome 1 (AtCry1) binds ATP and exhibits autokinase activity that is simulated by blue light. Similarly, it was reported that human cryptochrome 1 (HsCry1) exhibited autophosphorylation activity under blue light. To test the generality of light stimulated kinase function of cryptochromes, we purified AtCry1, AtCry2, HsCry1, and HsCry2 and probed them for kinase activity under a variety of conditions. We find that AtCry1, which contains near stoichiometric amounts of FAD and human HsCry1 and HsCry2 (which contain only trace amounts of FAD), has autokinase activity, but AtCry2, which also contains stoichiometric amounts of FAD, does not. Finally, we find that the kinase activity of AtCry1 is not significantly affected by light or the redox status of the flavin cofactor.     

Aqueous two-phase systems for molecular weight analyses

Summary The potential of aqueous two-phase systems for the estimation of molecular weights of proteins and enzyme-inhibitor complexes has been demonstrated. The method is simple and rapid, and employs extremely small amounts of the test sample. It is based on the measurement of the phase transition behaviour on dilution of the system as a function of the molecular weight of the molecule or complex under study.     

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G-3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.     

ROLE OF ALLELOPATHY AS EXPRESSED BY DOMINATING TREES IN A LOWLAND FOREST IN CONTROLLING THE PRODUCTIVITY AND PATTERN OF HERBACEOUS GROWTH

Low productivity rate and relatively bare areas occur under sycamore, hackberry, red oak, and white oak trees, even though several herbaceous species may grow well under elm trees in the same community which cast just as dense shade. No significant differences were found in amounts of most mineral elements and pH sampled under sycamore, hackberry, red oak, and white oak trees as compared with control soils under elm trees in the adjacent plots. Percent soil moisture was consistently higher under all test trees than under elm trees throughout the growing season. Thus the low productivity rate and relatively bare areas under sycamore, hackberry, red oak, and white oak trees were not due primarily to the factors stated above. Decaying leaves, leaf leachate of all four test species, and soil collected from under test trees significantly reduced seed germination, radicle growth, and seedling growth of selected herbaceous species. Several growth inhibitors, chiefly phenolics, were isolated and identified from sycamore, hackberry, red oak, and white oak leaves and the soils under them. Thus it appears that the low productivity rate and destitute growth under test tree species are due to allelopathy. Ecological significance of allelopathy in a community is discussed.     

Spatial releasing properties and mosquito repellency of cellulose-based beads containing essential oils and vanillin

Porous cellulose beads (Viscopearl) manufactured from wood pulp can provide gradual-release action for aromatic substances. Here we immersed lemongrass oil, xanthoxylum oil, and vanillin to apply mosquito repellents. The volatiles from this Viscopearl were analyzed to obtain information for quality control (QC) or specification using a GC–MS, and its standard compounds were determined as linalool, geranial, neral, and vanillin. In a test using a 20 L chamber, it was confirmed that their constant amounts were released continuously and they did not be affected by light. In another monitoring test for 47 days on the Viscopearl equipped with a commercialized air conditioner operated for 8 h every day under indoor conditions, the amounts of all released components increased in proportion to the open number of the pores on the module, which is a housing case containing the Viscopearl. In addition, the concentration of 8 major components including 3 standard QC compounds (linalool, geranial, and neral) in a repellency test room depended on both the ventilation and elapsed time. The vapor from the module did not affect main 7 plastic units of the air conditioner, whereas the Viscopearl equipped with an air conditioner showed good mosquito repellency under test chamber and room conditions that temperature, relative humidity, and carbon dioxide concentration were controlled at 24.8–25.4 °C, 63.8–65.7%, and 708–1383 ppm, respectively. Therefore, the plant essential oil-based repellent Viscopearl has high potential to be used as human protective agent against indoor mosquitoes by applying it to air conditioners.     

A mixture model approach to detecting differentially expressed genes with microarray data

An exciting biological advancement over the past few years is the use of microarray technologies to measure simultaneously the expression levels of thousands of genes. The bottleneck now is how to extract useful information from the resulting large amounts of data. An important and common task in analyzing microarray data is to identify genes with altered expression under two experimental conditions. We propose a nonparametric statistical approach, called the mixture model method (MMM), to handle the problem when there are a small number of replicates under each experimental condition. Specifically, we propose estimating the distributions of a t -type test statistic and its null statistic using finite normal mixture models. A comparison of these two distributions by means of a likelihood ratio test, or simply using the tail distribution of the null statistic, can identify genes with significantly changed expression. Several methods are proposed to effectively control the false positives. The methodology is applied to a data set containing expression levels of 1,176 genes of rats with and without pneumococcal middle ear infection.     

The liberation of amino acids and reducing compounds by plant roots

Summary Drying and remoistening of different plants grown in sand and in a sand-soil mixture resulted in the liberation of appreciable amounts of amino acids and detectable amounts of reducing compounds. Small quantities of amino acids were produced even under normal conditions of growth. The amino acids produced could be utilized for growth by amino acid requiring bacteria isolated from soil.Peas grown under aseptic conditions also yielded small amounts of ninhydrin-positive substances. Wheat under similar conditions produced less of such materials but in addition liberated detectable amounts of a reducing compound.These results are discussed in relation to the incidence of amino acid requiring bacteria in the rhizosphere.Contribution No. 385     

Human Ile-Ser-bradykinin, identical with rat T-kinin, is a major permeability factor in ovarian carcinoma ascites

Ascites from patients with metastatic ovarian carcinoma contains high amounts of an activity that increases vascular permeability, as easily detected by a rat skin test. Ascites was fractionated by gel permeation and reversed-phase high-performance liquid chromatographies. The fractions were analysed for permeability-increasing activity. In this way a peptide was isolated and identified as Ile-Ser-bradykinin by sequence and amino-acid analyses. It is identical with T-kinin which has previously been detected as a product of an acute-phase protein, T-kininogen, in rats but never in human material. The so far identified human kininogens, i.e. high- and low-molecular mass kininogens, can only release Met-Lys-bradykinin or its degradations products, as Ile-Ser-bradykinin is not a part of their structure. However, the present results provide evidence that the permeability factor Ile-Ser-bradykinin under certain conditions can be produced in considerable amounts also by human tissues.     

A straight-forward method of optimising protein solubility for NMR

Maximising solubility is a key step in applying solution-state NMR to proteins. The microbatch crystallisation screening method can be adapted to screen for protein solubility. In this approach, drops of test solutions are placed under paraffin oil in 96-well screening plates. This requires very small amounts of protein, is easy to set up and is readily automatable.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.     

长治市幼儿园4—5岁儿童四季生长情况

作者对山西省长治市幼儿园1980和1981年出生的216名儿童(男125、女91),在1985年四季生长的情况进行了测量和统计分析。结果表明,男童年均身高增长6.12厘米,体重增长2.49公斤;女童年均身高增长5.65厘米,体重增长2.35公斤。各季身高和体重增长值经F检验,季节差异均非常显著(p<0.01)。一年内最佳生长季节为秋季,同其它三季比较,经Dunnett检验,只有春季男女儿童身高增长和冬季女童体重增长与秋季相比,差异不显著(P<0.05)。其余各季增长同秋季比较,差异均非常显著(P<0.01)。     

Continuous ethanol production by zymomonas mobilis on an industrial scale

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was lower in the presence of oxygen than under anaerobic conditions. Aerobically, considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide. This bacterium contains considerable amounts of pentacyclic triterpenoids, mainly 1, 2, 3, 4-tetrahydroxypentane-29-hopane. It seems that stability and permeability of the cytoplasmic membrane of this rather ethanol tolerant organism is achieved by the hopanoid content. A continuous culture of Zymomonas mobilis produced 60 g/l ethanol over a test period of 39 days. This strain was used for ethanol production from an enzymatically hydrolyzed wheat starch fraction on an industrial scale of 100 m³.     

A novel and simple method of screening compounds for interaction with DNA: A validation study

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.     

Optimized group sequential study designs for tests of genetic linkage and association in complex diseases

The study of genetic linkage or association in complex traits requires large sample sizes, as the expected effect sizes are small and extremely low significance levels need to be adopted. One possible way to reduce the numbers of phenotypings and genotypings is the use of a sequential study design. Here, average sample sizes are decreased by conducting interim analyses with the possibility to stop the investigation early if the result is significant. We applied optimized group sequential study designs to the analysis of genetic linkage (one-sided mean test) and association (two-sided transmission/disequilibrium test). For designs with two and three stages at overall significance levels of.05 and.0001 and a power of.8, we calculated necessary sample sizes, time points, and critical boundaries for interim and final analyses. Monte Carlo simulation analyses were performed to confirm the validity of the asymptotic approximation. Furthermore, we calculated average sample sizes required under the null and alternative hypotheses in the different study designs. It was shown that the application of a group sequential design led to a maximal increase in sample size of 8% under the null hypothesis, compared with the fixed-sample design. This was contrasted by savings of up to 20% in average sample sizes under the alternative hypothesis, depending on the applied design. These savings affect the amounts of genotyping and phenotyping required for a study and therefore lead to a significant decrease in cost and time.     

Usefulness of single nucleotide polymorphism data for estimating population parameters

Single nucleotide polymorphism (SNP) data can be used for parameter estimation via maximum likelihood methods as long as the way in which the SNPs were determined is known, so that an appropriate likelihood formula can be constructed. We present such likelihoods for several sampling methods. As a test of these approaches, we consider use of SNPs to estimate the parameter Theta = 4N(e)micro (the scaled product of effective population size and per-site mutation rate), which is related to the branch lengths of the reconstructed genealogy. With infinite amounts of data, ML models using SNP data are expected to produce consistent estimates of Theta. With finite amounts of data the estimates are accurate when Theta is high, but tend to be biased upward when Theta is low. If recombination is present and not allowed for in the analysis, the results are additionally biased upward, but this effect can be removed by incorporating recombination into the analysis. SNPs defined as sites that are polymorphic in the actual sample under consideration (sample SNPs) are somewhat more accurate for estimation of Theta than SNPs defined by their polymorphism in a panel chosen from the same population (panel SNPs). Misrepresenting panel SNPs as sample SNPs leads to large errors in the maximum likelihood estimate of Theta. Researchers collecting SNPs should collect and preserve information about the method of ascertainment so that the data can be accurately analyzed.     

Sensitivity and specificity of a monitoring test

The usefulness of a diagnostic test is generally assessed by calculating the sensitivity and specificity, or the predictive value positive and predictive value negative of the test. When subjects are monitored periodically for evidence of disease, these calculations must incorporate the varying amounts of information per individual. If in addition, the test results lie on a continuous scale, these quantities vary with the cutoff value (cutpoint) used to define a positive test. They are usually calculated for a spectrum of potential cutpoints in order to produce receiver-operator characteristic curves. In this paper we use a partial likelihood solution to the discrete logistic model in order to obtain estimates of the diagnostic test indices and to provide a significance test when the diagnostic test is administered repeatedly to individuals.     

Studies on the production of anti-human-lymphocyte serum (ALS) in bulls]

The applicability of bulls as productive animals was considered for the preparation of anti-humans ALS. The course of immunologic response was studied by lymphoagglutination, lymphocytotoxicity, rosette inhibition, hemagglutination tests and by precipitin formation in two experimental groups immunized by different amounts of lymphocytes from peripheral blood of normal donors. The animals were found to respond well already after the second application of very small amounts of antigen (on day 0-4 times 10(7), on day 21-2 times 10(8) lymphocytes). They showed lymphoagglutination titre 1 : 512-2000, lymphocytotoxic titre being higher than 1 : 4000 and the rosette inhibition test gave a minimum titre of 1 : 65000. On the other hand, further application of a high amount of antigen (2 times 10(9), or 4 times 10(9) lymphocytes) did not lead to further increase in the titre; on the contrary - hyperimmunization resulted in a lower titre in the case of the rosette inhibition test, which is known to correlate best with the in vivo immunosuppressive activity. The hemagglutinin titre was also acceptable under the above conditions and the formation of undersirable precipitins against human serum proteins was negligible. Good response reached by a simple and economical immunization scheme speaks for the suitability of bulls for the production of ALS.     

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.     

Influence of kinship and spatial density on reconciliation and grooming in rhesus monkeys

Some cercopithecine primates direct disproportionate amounts of grooming, huddling, and agonistic support toward maternal kin. Disproportionate amounts of aggression are also directed toward maternal kin, however, suggesting that mechanisms that restore relationships damaged by aggression, such as reconciliation, might be biased toward these preferred social partners. Studies investigating kinship effects and reconciliation are inconsistent, however, perhaps because of differences in the environmental conditions under which behavior was observed. In order to test the effects of kinship and spatial density on affiliative and reconciliation behavior, we conducted focal and scan sampling on a group of rhesus monkeys (Macaca mulatta) living in an outdoor corral under low spatial density conditions. We then compared this data to previously published data on a group of the same species living under higher spatial density conditions. Neither overall grooming nor reconciliation were affected by spatial density once correction procedures were applied. Grooming was kin biased at both study sites, whereas reconciliation was kin biased only in the low-density group. Although data failed to support a Coping Model according to which grooming and reconciliation should go up under higher densities, we suggest that coping may be reflected not so much in overall rates of behavior but in strategic partner choices, such as the increased importance monkeys under crowded conditions appear to attach to nonkin partners. © 1996 Wiley-Liss, Inc.     

Quantitative determination of acylphosphatase levels in horse tissues by enzyme-linked immunosorbent assay

A non competitive enzyme-linked immunosorbent assay (ELISA) specific for horse muscle acylphosphatase (E.C. 3.6.1.7.) has been developed. The purified anti-acylphosphatase antibodies were immobilized by passive absorption to a solid-phase support and incubated with known and unknown amounts of antigen. The antibody-acylphosphatase complex was quantified using the same antibody conjugated to horseradish peroxidase. The assay yields positive reactions with as little as 0.05 ng of antigen, with intra- and interassay coefficients of variation of 5% and 7%, respectively. On the basis of this assay we developed a more sensitive test than the optical one, using benzoyl-phosphate as substrate, for acylphosphatase determination. By means of this test, the presence of the enzyme in horse tissue homogenates was evaluated under conditions in which the optical test failed to distinguish the acylphosphatase activity from that of other enzymes.