Intra-chromosomal position interference inNeurospora crassa

Multiple-point crosses where 20 pairs of regions (ten loosely linked markers) for the study of contiguous exchanges involving two linkage groups, capable of being investigated at a time, were utilized. In order to find out the effect of a chelating agent on interference, crosses were treated with different molar concentrations of ethylene-diamine tetraacetic acid (EDTA). All marker strains were standardized before use by inbreeding with a wild-type of known parentage.Since tests based on Coefficient of Coincidence and on Poisson distribution for finding out the position interference are rather unsatisfactory, a method based onContingency Chi-square test for detecting the intensity and nature of interference is described.Data obtained from 1813 analyzable ordered tetrads show that positive interference is absent in the control crosses. It is present among certain regions in crosses when treated with 4×10^–5M and 10×10^–5M EDTA but it vanishes again in crosses when treated with 20×10^–5M EDTA. Negative interference is present in the control crosses but it varies among pairs of regions on the two linkage groups. The localization and intensity of interference are alterable with EDTA treatment. The data are discussed in the light of certain concepts invoking to explain the mechanism that involves a genetic exchange.     

Increased acidophilia of eosinophil granules after EDTA treatment

Summary The acidophilic reaction of eosinophil leucocyte granules from human, pig and horse blood smears was investigated by using May—Grünwald—Giemsa staining after previous treatment with EDTA and sodium citrate solutions. The same peak at 530 nm, but absorption values considerably higher than those of controls, were found in eosinophil granules after application of chelating agents, indicating that removal of metal cations could unmask basic groups in these structures.     

The Effect of Chelating Agents on Soil Sodicity

Results of laboratory and field tests suggest that chelating agents could be used to alleviate adverse soil properties caused by excess sodium, such as low permeability. Adding multi-dentate carboxylic acid chelating agents to sodic soil, or to mixtures of soil with sodium-contaminated waste, significantly reduced sodium adsorption ratio (SAR) values. Judging from cation concentrations in saturated paste (sat. paste) filtrates, chelating agents act to ameliorate soil sodicity by releasing Ca and to a lesser extent Mg from undissolved compounds. After adding chelating agents to moist soils that contained free lime, measured weight losses were consistent with CO ₂ evolution due to CaCO ₃ decomposition. The electrical conductivity (EC) of the sat. paste filtrate of materials treated with chelating agents increased less than when equivalent Ca or Mg was supplied in conventional, soluble form. Bigger sat. paste vacuum filtration volumes, improved soil permeability and faster field infiltration rates were observed after treatment with chelating agents. The Ca- and Mg-complexes of agents such as citric and malic acid degrade in moist soil; such agents could perhaps be used in a series of applications to improve ease of cultivation and permeability of cropped land. The agent ethylenediamine tetraacetic acid (EDTA) forms stable complexes, and could therefore be used as a one-time treatment for sodic materials that are to be disposed of by burial, following guidelines for soil SAR and EC.     

Effect of Chelate-Assisted Hexavalent Chromium on Physiological Changes,Biochemical Alterations,and Chromium Bioavailability in Crop Plants—An In Vitro Phytoremediation Approach

This study revealed heavy metal–induced physiological and biochemical alterations in crop seedlings by supplementing chelating agents in the nutrient solution. Hexavalent chromium (Cr⁺⁶) induces several toxic effects in hydroponically grown rice, wheat, and green gram seedlings. A noticeable decrease was observed in root length, shoot length, biomass content, and chlorophyll biosynthesis of the seedlings grown in the nutrient solutions supplemented with Cr⁺⁶ at 100 μM. The seedling growth was stimulated with supplement of chelating agents such as EDTA, DTPA, and EDDHA. An increase in proline content was noticed with the application of Cr⁺⁶ (100 μM) in nutrient solutions. Stimulated activities of antioxidant enzymes such as catalase and peroxidase were noticed with increasing concentrations of chromium. Cr bioaccumulation was significantly high in roots of seedlings treated with Cr⁺⁶ at 100 μM in nutrient solution. Shoot translocation of Cr as depicted by transportation index (Ti) values for different crops were enhanced with the application of chelating agents. The total accumulation rate (TAR) for Cr was enhanced with the supplementation of DTPA in rice and wheat, whereas the application of EDDHA was found effective for increasing the accumulation rate of Cr in green gram seedlings. This study demonstates the role of chelating agents in lessening the toxic effects of Cr⁺⁶. The chelating agents supplemented with Cr⁺⁶ in the culture medium enhanced the Cr bioavailability in plants.     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

INCOMPATIBILITY IN AMSINCKIA GRANDIFLORA (BORAGINACEAE): DISTRIBUTION OF CALLOSE PLUGS AND POLLEN TUBES FOLLOWING INTER- AND INTRAMORPH CROSSES

The distribution of callose plugs and pollen tubes was investigated following inter- and intramorph crosses of Amsinckia grandiflora (Boraginaceae), a distylous species possessing cryptic self-incompatibility. Callose plug distribution provided a good indication of the distribution of pollen tubes. Compared to intramorph crosses, many more callose plugs and pollen tubes were found in basal stylar regions following intermorph crosses, indicating that differential pollen tube growth is a likely cause of cryptic self-incompatibility. The incompatibility response differed for the floral morphs: in the pin (long-styled) morph pollen tubes were most likely to cease growth in the midstylar region, while inhibition was more likely to occur in the upper stylar region of the thrum (short-styled) morph. There was no evidence of stigmatic inhibition of pollen tubes for either morph, although the incompatibility response in the Boraginaceae is normally located in the stigmatic region.     

Sensitivity of bacterial coaggregation to chelating agents

Coaggregation between pairs of microorganisms was found to be inhibited by chelating agents, such as acetylacetone, citrate, EDTA and carboxymethylcellulose. Assays were conducted on eight pairs of periodontopathogens and one pair consisting of Escherichia coli and Saccharomyces cerevisiae. The inhibitory effects of the chelating agents were reversible except for Actinomyces naeslundii 12104, the adhesin of which was irreversibly inactivated. Even though the bacteria possessed different kinds of adhesins, their sensitivity to chelating agents appears to be a common property. Non-toxic chelating agents, such as carboxymethylcellulose and citrate, may prove to be useful anti-adhesins.     

Effect of divalent cations and chelators on metaphase to telophase progression and nuclear envelope formation in Chinese hamster cells

Chinese hamster DON cells in log phase were treated with Colcemid in the G₂ period with or without divalent cation chelating agents. The metaphase cells were isolated and incubated in two ways: 1) without Colcemid but with chelating agents or La³⁺ and observed for metaphase to telophase progression, and 2) with Colcemid, with or without chelating agents and the rate of micronuclei formation in the absence of anaphase monitored. The effect of the chelating agents on cellular ⁴⁵Ca²⁺ during metaphase to telophase progression was also studied.The results indicate that Ca²⁺ and possibly Mg²⁺ ions are involved in the regulation of certain segments of mitosis. The reduction of environmental and plasma membrane associated Ca²⁺ with the chelators and La³⁺ promoted the metaphase to telophase progression as well as nuclear envelope and micronuclei formation.     

Crossing over and Diploid Egg Formation in the Elongate Mutant of Maize

Rhoades (1941) found recombination in the proximal regions of chromosome 5 to be higher in male than in female flowers. Two explanations were proposed to account for the lower female values, namely: (1) there is a basic difference in rates of crossing over in mega- and microsporocytes, or (2) selective orientation of the chromosome 5 bivalent on the meiotic spindle leads to the preferential segregation of noncrossover chromatids to the basal megaspore. These alternatives have been tested by carrying out a half-tetrad analysis of the diploid eggs produced by plants homozygous for the recessive elongate (el) allele. The A2-Bt crossover values determined from the diploid eggs of elongate plants were much lower than those calculated from haploid sperm of both El el and el el plants. Since male and female flowers should have similar cross-over values if the orientation hypothesis were correct, it was concluded that the amount of crossing over in the A2-Bt region of chromosome 5 is intrinsically higher in male than in female meiocytes. In the analysis of diploid eggs the use of the Bt locus, which marks the centric region of chromosome 5, provided information on the origin of diploid eggs. The genotypic constitution of 425 diploid eggs was ascertained. Of these, 20.4% were Bt bt. They could not be accounted for by failure of the second meiotic division or by replication during the interphase between the two meiotic divisions, but are expected if there is a single division with an equational separation of the centromere regions of chromosome 5. The Bt Bt and bt bt genotypes arise from a disjunctional separation. It is proposed that diploid eggs are produced by an abnormal meiosis in which there is one division with either disjunctional or equational separation. Disjunctional separation is more frequent but the ratio of the two types varies from ear to ear. Recombination in the A2-Bt-Pr region of chromosome 5 was found to be higher in the haploid gametes of elongate homozygotes than in El El and El el plants. On the other hand, crossing over was reduced in the Sh-Bz segment of chromosome 9 in elongate plants, but the adjacent Bz-Wx interval was unaffected.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams

This study investigates the hypothesis that loosening of the egg-box structure by presence of divalent ion chelating agents during enzymatic degradation of homogalacturonan (HG) can improve enzymatic polysaccharide solubilization on pectinaceous, agro-industrial co-processing streams. The influence of different levels of ethylene-diaminetetraacetic acid (EDTA), citric acid, oxalic acid, and phosphate was assessed in relation to enzymatic solubilization of isopropanol precipitatable oligo- and polysaccharides from sugar beet pulp, citrus peel, and two types of potato pulp. The two types of potato pulp were FiberBind 400, a dried commercial potato pulp product, and PUF, a dried calcium reduced product, respectively. The enzymatic treatment consisted of 1% (w/w) of substrate treated with pectin lyase from Aspergillus nidulans and polygalacturonase from A. aculeatus [each dosed at 1.0% (w/w) enzyme/substrate] at 60 °C, pH 6.0 for 1 min. Characterization of the released fractions demonstrated a significantly improved effect of chelating agents for polysaccharide solubilization from FiberBind 400, PUF, and citrus peel, whereas only low amounts of polysaccharides were solubilized from the sugar beet pulp. The results substantiated the importance of chelating agents during enzymatic extraction of pectinaceous polysaccharides. Lower levels of chelating agents were required for the calcium-reduced potato pulp substrate (PUF) indicating the significance of calcium cross-linking in HG in relation to the enzymatic solubilization yields. The effect of the chelating agents correlated to their dissociation constants (pK_a values) and calcium binding constants and citric acid and EDTA exerted highest effects. Maximum polysaccharide yield was obtained for FiberBind 400 where the enzymatic treatment in presence of citric acid yielded 22.5% (w/w) polysaccharides of the initial substrate dry matter.     

Iron and iron chelating agents modulate Mycobacterium tuberculosis growth and monocyte-macrophage viability and effector functions

Excess of iron promotes Mycobacterium tuberculosis infection, its replication and progression to clinical disease and death from tuberculosis. Chelation of iron may reduce M. tuberculosis replication, restore host defence mechanisms and it could constitute an application in the prevention and treatment strategies where both iron overload and tuberculosis are prevalent. We investigated the effect of iron and iron chelating agents, like desferrioxamine and silybin, individually and in combination with iron on mycobacterial number, viability in culture and after recovery from monocyte-macrophages, together with monocyte-macrophages viability and oxidative defence. Mycobacterial number and viability in culture were assessed using real-time quantitative PCR of H37Rv IS6110 DNA, 16S rRNA and 85B mRNA, whereas the microplate AlamarBlue(TM) assay was used to detect viability in culture post-infection. Mitochondrial membrane potential and phosphatidyl serine exposure of monocyte-macrophages, detected using Mitotracker Red fluorescence and Annexin V binding, respectively, served as indicators of host cell viability. Superoxide generation served as marker of monocyte-macrophage effector functions. Extracellular H37Rv showed a significant increase in number and viability in presence of excess iron and, by large, a significant decrease in number and viability in presence of the iron chelating agents, silybin and desferrioxamine, compared to cultivation without supplementation. Intracellularly, excess iron increased H37Rv viability significantly but reduced monocyte-macrophages mitochondrial membrane potential and compromised superoxide production. Desferrioxamine had little influence on intracellular parameters, but consistently prevented effects of excess iron, while silybin significantly altered most intracellular parameters and mostly failed to prevent effects of excess iron. These findings suggest that chelation therapy should be considered in conditions of iron overload and that effective chelating agents like desferrioxamine, with limited intracellular access might need to be used in combination with lypophilic chelating agents.     

Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase.

In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.     

The catalytic properties of a proteinase isolated from sheep abomasal mucosal mast cells

The catalytic properties of a sheep mast cell proteinase (SMCP), isolated from abomasal mucosal mast cells, were investigated. The enzyme was shown to have chymotrypsin-like esterase activity, with no detectable amide activity, using a range of low molecular weight substrates. Maximal activity, against Benzyloxycarbonyl-L-tyrosine-4-nitrophenol ester, was determined to be in the range pH 7.6-8.0. Inhibitor studies showed that, unlike chymotrypsin, a serine proteinase, SMCP was found to be susceptible to the action of thiol blocking agents and chelating agents, but to be unaffected by diisopropylphosphofluoridate, a serine proteinase inhibitor.     

Cadmium inhibits delta-aminolevulinate dehydratase from rat lung in vitro: interaction with chelating and antioxidant agents

The effect of cadmium (Cd(2+)) on delta-aminolevulinate dehydratase (delta-ALA-D) activity from rat lung in vitro was investigated. delta-ALA-D activity, a parameter for metal intoxication, has been reported as a target of Cd(2+) in different tissues. The protective effect of monotherapies with dithiol chelating (meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS)) or antioxidant agents (ascorbic acid, diphenyl diselenide (PhSe)(2), and N-acetylcysteine (NAC)) was evaluated. The effect of a combined therapy (dithiol chelatingxantioxidant agent) was also studied. Zinc chloride (ZnCl(2)) and dithiothreitol (DTT) were used to investigate the mechanisms involved in cadmium, chelating and antioxidant effects on delta-ALA-D activity. Cadmium inhibited rat lung delta-ALA-D activity at low concentrations. DTT (3mM), but not ZnCl(2) (100microM), protected the inhibition of enzyme activity caused by Cd(2+). Chelating agents were not effective in restoring the enzyme activity. DMPS and DMSA presented inhibitory effect on enzyme activity. DTT restored the inhibition caused by both chelating agents, but ZnCl(2) restored only the inhibitory effect induced by DMSA. These compounds caused a marked potentiation of delta-ALA-D inhibition induced by Cd(2+). ZnCl(2) did not restore inhibition of enzyme activity caused by Cd(2+) plus chelating agents. Conversely, DTT restored the inhibition induced by Cd(2+)/DMSA, but not by Cd(2+)/DMPS. Antioxidants were not effective in ameliorating delta-ALA-D inhibition induced by Cd(2+), whereas ascorbic acid potentiated the enzyme inhibition induced by this metal. A combined effect of Cd(2+)xDMPSx(PhSe)(2) and Cd(2+)xDMPSxNAC was observed. There was no combined effect of Cd(2+)xchelatorxantioxidants when DMSA was used. This study demonstrated that Cd(2+)inhibited delta-ALA-D activity and chelating and antioxidant agents, alone or combined, did not restore the enzyme activity. In contrast, these compounds potentiated the inhibition induced by Cd(2+) in rat lung.     

Region-Specific Recombination in Phage T4. II. Structure of the Recombinants

In this paper, we present results of crosses designed to elucidate the structure of recombinants in the tail-fiber region of bacteriophage T4, in which a glucosylation-dependent recombinations mechanism is operative, and the cause of the "special" recombination in glycosylated crosses is discussed. We present evidence that, when phage are nonglycosylated, recombination in the tail-fiber region proceeds via long heteroduplex overlaps. Mismatched bases within such regions (in nonglycosylated phage) are repaired efficiently (as contrasted to those of glucosylated phage), but asymmetrically; that is, there may be an equal probability of resolving the mismatch to mutant or wild type.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Recombination around the Tm2a and Mi resistance genes in different crosses of Lycopersicon peruvianum

The amount of recombination in three different intraspecific crosses of the wild tomato species Lycopersicon peruvianum was investigated for the short arm of chromosome 6 that harbors the Mi nematode resistance gene and the centromeric region of chromosome 9 that contains the Tm2a virus resistance gene. These two genes have been introgressed into the cultivated tomato and are associated with a significant reduction in recombination in the respective region when crossed to other L. esculentum lines. For both regions and all crosses within L. peruvianum significantly more recombination (up to more than ten fold) was observed in the gametes derived from the female parent than in those from the male parent. In general, the differences were more pronounced for chromosome 6 than for chromosome 9. The amount of recombination in the three intraspecific L. peruvianum crosses was compared with the amount of recombination observed in the standard interspecific cross used for the construction of a saturated genetic map of tomato (L. esculentum x L. pennellii). In two of three cases for each region, more recombination was observed in the intraspecific crosses and in one case for each region significantly less recombination was found in the intraspecific cross when compared to the interspecific cross. Specifically for the Mi-carrying region, crosses within L. peruvianum exhibited up to 15-fold more recombination than crosses between resistant and susceptible L. esculentum lines, and such crosses will allow the fine mapping of this gene for the purpose of map-based cloning.     

Control of glutamate dehydrogenase from Pisum sativum roots

-Glutamate dehydrogenase (GDH) was found in soluble and particulate (mitochondrial) fractions of pea roots. The activity of NADH-dependent GDH in fresh mitochondrial extract was increased about 10-fold by addition of zinc, manganese or calcium, but high concentrations of zinc were inhibitory. During storage, GDH activity of the mitochondrial extract slowly increased. The NADH activity was inhibited by citrate and other chelating agents. NADH-dependent reductive amination was also inhibited by glutamate, the product of the reaction; by contrast NADPH dependent activity was relatively unaffected by zinc, chelating agents or glutamate. Sensitivity (of NADH-GDH) to glutamate was lost on purification, but was restored when the enzyme was immobilized by binding to an insoluble support (AE cellulose). Glutamate appears to change the affinity of the enzyme for 2-oxoglutarate.