Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK^+/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.     

Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TK+/− mutation,DNA single-strand break,and Balb/c 3T3 chemical transformation assays

The activity of quercetin was investigated in (a) the L5178Y TK^+/− mutation assay system, using trifluorothymidine (TFT) as the selection agent; (b) the DNA single-strand break assay in L5178Y cells after the same treatment used for the mutation assay; and (c) the Balb/c 3T3 chemical transformation assay (foci method). Quercetin was active in the TK^+/− mutation assay, increasing the frequency of TFT-resistant colonies from a control value of 37 per 10⁶ viable cells to 355 per 10⁶ viable cells at 20 μg/ml. When S9 was present, the activity was decreased at each concentration tested. As the S9 concentration employed (mg/ml protein) was decreased, the induced mutant frequency increased. DNA single-strand breakage was observed without S9 at 10 μg/ml, using the alkaline elution technique; a maximal rate of elution was reached at 20 μg/ml. In the chemical transformation experiments, transformation just at the level of 0.05% significance (if both intermediate and typical transformed colonies were combined) was observed. The evidence is sufficiently strong that additional attention should be given to its role as a dietary caused of human cancer.     

The utilization of trifluorothymidine (TFT) to select for thymidine kinase-deficient (TK−/−) mutants from L5178Y/TK+/− mouse lymphoma cells

Trifluorothymidine (TFT), a thymidine analog, was analyzed for its ability to select for thymidine kinase-deficient (TK^−/−) mutants. In comparison with BUdR, the traditional selective agent for TK^−/− cells, it was determined that TFT at 1/50th the dose (1μg/ml vs. 50 μg/ml) is a more effective and versatile selective agent for TK^−/− mutants arising from the TK^+/−-3.7.2C heterozygote of L5178Y mouse lymphoma cells. Since TFT acts more rapidly than BUdR, it can be utilized in procedures (such as the analysis of the phenotypic lag) requiring the fast arrest of cell division. Reconstruction analyses of effective TK^−/− mutant recovery indicate that TFT can be used to recover mutants from significantly higher densities of TK^+/− cells than can BUdR. In addition, TK^−/− mutants can attain larger colony size in TFT than in BUdR where severe stunting of growth occurs at high TK^−/− cell densities. 190 of 194 isolated TFT-resistant large and small colony mutants (both spontaneous and induced)     

Nuclear translocation of the p65 subunit of NF-κB in L5178Y sublines differing in antioxidant defense

We examined the induction of nuclear translocation of the p65 subunit of NF-κB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-κB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-κB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 μM, 24 h, 37°C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-κB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells. Received: 10 November 1998 / Accepted in revised form: 2 March 1999     

Disulfide bonds Cys9–Cys57, Cys34–Cys88 and Cys38–Cys90 of the β-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the α-subunit: experimental evidence for the conclusions from the crystal structure of hCG

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α- and β-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-β in heterodimer formation with the α-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-β were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of α- and β-subunits. The disulfide peptides Cys (9–57), Cys (34–88) and Cys (38–90) were found to inhibit the α/β recombination whereas the remaining three disulfide peptides viz. Cys (23–72), Cys (26–110) and Cys (93–100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the α/β recombination. Results clearly demonstrate that the disulfide bonds Cys⁹–Cys⁵⁷, Cys³⁴–Cys⁸⁸ and Cys³⁸–Cys⁹⁰ of the β-subunit of hCG are crucial for heterodimer formation with the α-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.     

A solid-phase immunosorbent assay to determine the proteinase binding capacity of α2-macroglobulin using 125I-trypsin as indicator proteinase

Hyperimmune sera against human α₂macroglobulin were raised in rabbits following immunization with ‘s’ α₂-macroglobulin⁷ over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for ‘s’ and ‘f’ α₂-macroglobulin (plasma α₂-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4·10^?6 as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, ‘s’ α₂-macroglobulin and both ‘f’ α₂-macroglobulins (27 and 82% saturation of ‘s’ α₂-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact α₂-macroglobulin subunit (M_r 182 000) with ¹²⁵I-labeled trypsin binding capacity of immunoimmobilized α₂-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between ‘s’ α₂-macroglobulin and ‘f’ α₂-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol α₂-macroglobulin was observed. Saturation of solid phase immunoimmobilized ‘s’ α₂-macroglobulin with trypsin could be accomplished by incubation with a 100–200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol α₂-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized α₂-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to α₂-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of α₂-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive α₂-macroglobulin of 0–53% when compared to the trypsin-binding capacity of normal plasma α₂-macroglobulin.     

Synthesis and evaluation of an 18F-labeled boramino acid analog of aminosuberic acid for PET imaging of the antiporter system xC−

In this study, we synthesized ¹⁸F-ASu-BF₃, a close boramino acid analog of 5-[¹⁸F]fluoro-aminosuberic acid (¹⁸F-ASu), via ¹⁸F-¹⁹F isotope exchange reaction and evaluated its potential for imaging with positron emission tomography (PET). ¹⁸F-ASu-BF₃ was stable in mouse plasma and taken up into PC3 prostate cancer cells via the system x_C^? amino acid transporter. The continuous use of isoflurane for anesthesia during dynamic imaging acquisition slowed down the excretion of ¹⁸F-ASu-BF₃ and enabled visualization of PC3 tumor xenografts in mice. In contrast, no tumor visualization was observed from static images of ¹⁸F-BF₃-ASu due to its rapid renal excretion mediated in part by the organic anion transporter. Our data indicate that the pharmacokinetics of amino acids could be altered after being converted into their boramino acid analogs. Therefore, care should be taken when using the boramino acid strategy to design and prepare ¹⁸F-labeled tracers for imaging amino acid transporters/receptors with PET.     

Issues in using the WinRHIZO system to determine physical characteristics of plant fine roots☆

Image analysis systems have facilitated rapid measurements of fine root length (RL), diameter (RD), volume (RV), etc. The WinRHIZO system is unlike other image analysis systems in that it can detect, and make corrections for, areas of root overlap. It is designed to be capable of using both Regent’s non-statistical method (WinRHIZO method) and Tennant’s statistical method (line-intersect method), and can simultaneously output the root measurements by both methods when they are chosen at the same time. This study tested: (1), the efficacy of the overlap correction function in the WinRHIZO system; and (2), the consistency of fine root measurements between the WinRHIZO and Tennant methods with two sets of root measurement data from winter wheat (Triticum aestivium L.). The results showed that there were significant differences in RL, RD and RV between small root samples with and without stumps. The impact of root stumps outweighed the overlap correction efficacy in WinRHIZO. The values from the Tennant method are significantly different from those using the WinRHIZO method, although both results are statistically closely correlated. This indicated how critical it was to use without-stump root samples when using image analysis systems to measure RL, RD, RV, etc., and to keep in mind that a significant difference in root measurements may be methodologically related when comparing the results of various experiments from these two methods. Our research results bear important implications for the study of root ecology.     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the -insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the -insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the -insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two -insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 M^–1 s^–1 respectively, compared to 10 and 2.5 M^–1 s^–1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the -insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶Ser can compensate somewhat for the loss of binding at the -insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the -insertion loop, thereby causing a decrease in binding efficiency.     

On the formation of rho? petites in yeast

Summary The inheritance of an extrakaryotic mutation conferring temperature-sensitive growth on nonfermentable substrates and a high frequency of mutation to rho^– has been studied. Multifactorial crosses (rho⁺xrho⁺) involving this mutation T ₈ ^S and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin or paromomycin revealed: a) Mutation T ₈ ^S is localized on the mitDNA, referring to a new gene locus TSM1. b) Locus TSM1 appears to be weakly linked to the locus PAR1 and to the loci RIB1 and RIB3 but unlinked to the locus OLI1. c) The position of TSM1 is between PAR1 and the two closely linked loci RIB1 and RIB3, OLI1 is outside and not linked to the segment PAR-TSM-RIB. d) Mutation T ₈ ^S does not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus .     

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.     

An assay for mutagenic activity of 4CMB, 4HMB and BC,using the “microtitre” fluctuation test

4CMB, 4HMB and BC were assayed for mutagenic activity using the ‘microtitre’ bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects base-substitution mutation. 4HMB was negative in both strains. 4CMB and 4HMB were equally toxic to the strains, whilst BC was comparatively less toxic.     

A comparative study of the mechanism of Na+ and Cl? excretion by the gill ofMugil capito andFundulus heteroclitus: Effects of Stress

Summary In seawater (SW)-adaptedMugil andFundulus, gill effluxes of Na⁺ and of Cl^– and the simultaneously recorded transgill potential (P.D.) differ according to whether they are measured in stressed or rested animals.In rested animals of the two species, transfer to Ringer's solution considerably reduces the P.D. but not . InFundulus, is also decreased. Transfer of the two species from SW to fresh water (FW) reduces and by 75 to 85% and leads to a large inversion of P.D. When K⁺ is added to FW, a gill depolarization occurs, as well as a large increase of and .These results suggest that: 1) the P.D. originates primarily from the diffusion of cations, the gill permeability to Na⁺ ( ) being greater than that to Cl^– ( ), 2) a Cl^–/Cl^– exchange independent of P.D. is associated with the Cl^– pump; 3) Cl^– pump activity is linked to Na⁺/K⁺ exchange which in turn is associated to a Na⁺/Na⁺ exchange diffusion mechanism.In stressed individuals of the two species, the P.D. in SW, as well as the P.D. changes observed during transfer experiments, are considerably reduced. The decrease of and observed after transfer from SW to FW are also minimised. Changes are smaller inFundulus. The decrease of P.D. characterizing stressed animals may be at least in part due to a 3 to 4 fold increase of which becomes equal to in both species.As a result of stress, the K⁺-activated Na⁺ and Cl^– excretion mechanisms are totally inhibited inFundulus and partially so inMugil.Stress response seems more intense inFundulus and recovery from stress faster inMugil.     

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta? 2 using a highly representative rice BAC library

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta ² (closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 ± 1.3 and 8.0 ± 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta ² region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a “two-step walking” method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta ² . The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta ² region showed various cross-hybridizing signals near the centromeric regions of all chromosomes. Received: 14 August 1996 / Accepted: 2 December 1996     

The nature of the Ln3+ —angiotensin II complex. A 13C nmr study of the binding of Yb3+ to angiotensin ii

The nature of the Yb³⁺-angiotensin II complex is examined by ^13C nuclear magnetic resonance. The ytterbium-induced shifts of most resonances are observed to be strongly dependent on pD, while a few are observed to be largely independent of pD. These observations are shown to be consistent with stepwise binding of the lanthanide ion to the carboxylates of aspartic acid and the C-terminus.     

An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

Some Bacillus thuringiensis strains secrete β-exotoxin, which is an insecticidal, thermostable adenine nucleotide analogue. Discrepancies between detection of β-exotoxin by high-performance liquid chromatography and insect bioassays have shown the importance of bioassays in the determination of β-exotoxin production. With the aim of improving the fly β-exotoxin bioassay, a range of fly diets were evaluated and the best performing diet was incorporated into a novel β-exotoxin bioassay. The improved bioassay is characterised by good control pupation percentages, low variability, easy setup and monitoring. The bioassay allowed unambiguous differentiation between β-exotoxin producing and non-producing strains, and is suitable for the routine screening of B. thuringiensis strains for β-exotoxins.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.