Studies on the biochemical basis of spontaneous mutation. V. Effect of temperature on mutation frequency

Temperature, in the range of 15 °C to 40 °C, has a pronounced effect on the incorporation of 2-aminopurine deoxynucleotides into DNA by purified bacteriophage T4-induced DNA polymerase. Whereas the total rate of utilization of the 2-aminopurine deoxynucleoside triphosphate increases with increasing temperature, a greater proportion is converted to the monophosphate by the editing nuclease of the enzyme. Therefore, the amount of analogue incorporated goes through a maximum and then decreases with increasing temperature. These results, obtained in vitro, have been correlated with effects of temperature on 2-aminopurine induced and spontaneous mutation rates of several rII markers, and they have been generalized to an hypothesis which holds that the stability of the helix immediately preceding the incoming nucleotide is an important factor in determining the accuracy of DNA replication. We suggest that there is a higher probability of making errors via base substitutions in a more stable (G + C-rich) rather than a less stable (A + T-rich) microenvironment.     

Studies on the biochemical basis of spontaneous mutation. III. Rate model for DNA polymerase-effected nucleotide misincorporation

We propose a model to investigate the relation between insertion and excision activities of polymerases involved in DNA synthesis, and the frequency of errors resulting from substituting either mismatched bases or base analogues into a DNA molecule. An analytical equation is derived which expresses the error frequency as a function of nucleotide insertion and removal rates. For the general case, given arbitrary rates of insertion and removal, and allowing the enzyme to peel back by excising previously incorporated nucleotides, we have developed a computer simulation for the synthesis of a DNA molecule. In the special case, where insertion and removal frequencies are within the biologically interesting range for spontaneous mutations, the effect of “peelback” on error correction can be obtained analytically. Our results suggest that the magnitude of the removal frequency (3′-exonuclease activity) is the parameter that exerts the greatest influence on error correction capability; the frequency of errors is less sensitive to either the specificity for removal of mismatched relative to correctly matched bases, or to peelback.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

Biochemical basis of ozone toxicity

Ozone (O3) is the major oxidant of photochemical smog. Its biological effect is attributed to its ability to cause oxidation or peroxidation of biomolecules directly and/or via free radical reactions. A sequence of events may include lipid peroxidation and loss of functional groups of enzymes, alteration of membrane permeability, and cell injury or death. An acute exposure to O3 causes lung injury involving the ciliated cell in the airways and the type 1 epithelial cell in the alveolar region. The effects are particularly localized at the junction of terminal bronchioles and alveolar ducts, as evident from a loss of cells and accumulation of inflammatory cells. In a typical short-term exposure the lung tissue response is biphasic: an initial injury-phase characterized by cell damage and loss of enzyme activities, followed by a repair-phase associated with increased metabolic activities, which coincide with a proliferation of metabolically active cells, for example, the alveolar type 2 cells and the bronchiolar Clara cells. A chronic exposure to O3 can cause or exacerbate lung diseases, including perhaps an increased lung tumor incidence in susceptible animal models. Ozone exposure also causes extrapulmonary effects involving the blood, spleen, central nervous system, and other organs. A combination of O3 and NO2, both of which occur in photochemical smog, can produce effects which may be additive or synergistic. A synergistic lung injury occurs possibly due to a formation of more powerful radicals and chemical intermediates. Dietary antioxidants, for example, vitamin E, vitamin C, and selenium, can offer a protection against O3 effects.     

The biochemical basis of mitochondrial diseases

Disfunctioning of human mitochondria is found in a rapidly increasing number of patients. The mitochondrial system for energy transduction is very vulnerable to damage by genetic and environmental factors. A primary mitochondrial disease is caused by a genetic defect in a mitochondrial enzyme or translocator. More than 60 mitochondrial enzyme deficiencies have been reported. Secondary mitochondrial defects are caused by lack of compounds to enable a proper mitochondrial function or by inhibition of that function. This may result from malnutrition, circulatory or hormonal disturbances, viral infection, poisoning, or an extramitochondrial error of metabolism. Once mitochondrial ATP synthesis decreases, secondary mitochondrial lesions may be generated further, due to changes in synthesis and degradation of mitochondrial phospholipids and proteins, to mitochondrial antibody formation following massive degradation, to accumulation of toxic products as excess acyl-CoA, to the depletion of Krebs cycle intermediates, and to the increase of free radical formation and lipid peroxidation.     

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity

Background

Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity.

Results

In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na⁺, K⁺ and Ca²⁺ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed.

Conclusions

The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs’ toxic actions.     

眼镜蛇神经毒素的急性毒性实验研究

目的　研究眼镜蛇神经毒素 （ Ｃｏｂｒａ ｎｅｕｒｏｔｏｘｉｎ, Ｎ Ｔ） 的急性毒性和蓄积毒性。方法　测 Ｎ Ｔ 对小鼠的 Ｌ Ｄ５０ ; 对大鼠、狗的１ 次性最小中毒剂量和最大安全剂量; 计算 Ｎ Ｔ 在小鼠、狗体内的２４ｈ 蓄积率。结果　 Ｎ Ｔ 经静注、肌注、腹腔、皮下 ４ 种途径给药对小鼠的 Ｌ Ｄ５０ 分别是 （１９５±９５） μｇ／ｋｇ、（１５６±８５） μｇ／ｋｇ、（１５１±１９） μｇ／ｋｇ、（１８４±８５） μｇ／ｋｇ, 对小鼠的最小致死剂量为９７５μｇ／ｋｇ。 Ｎ Ｔ 对大鼠、狗的１ 次性中毒剂量分别为５４μｇ／ｋｇ 和３４μｇ／ｋｇ。对小鼠、大鼠和狗的安全剂量分别为８１５μｇ／ｋｇ、４２μｇ／ｋｇ和３０μｇ／ｋｇ, 分别约为人临床用剂量 （７０μｇ／５０ｋｇ·ｄ－ １ ） 的５８２、３０ 和２１ 倍。 Ｎ Ｔ 在小鼠、狗体内的２４ｈ蓄积率分别为 ５７％ 和３０％ 以上。结论　 Ｎ Ｔ 在使动物中毒的剂量下有广泛的安全范围; Ｎ Ｔ 在动物体内存在弱蓄积毒性。     

On the biochemical basis of hereditary fructose intolerance.

In hereditary fructose intolerance it was found that in addition to an increased K_m value for Fru-1-P, the K_m of aldolase for Fru-1,6-P₂ was also increased. Furthermore, human phosphorylase $\text{a}$ was found to be inhibited by Fru-1-P in a non-competitive way.     

Studies on the biochemical basis of mutation VI. Selection and characterization of a new bacteriophage T4 mutator DNA polymerase

A new mutant of bacteriophage T4 has been isolated by a procedure which was designed to select for mutants with high spontaneous reversion rates. This mutant, M19, induces a defective DNA polymerase which has a degraded specificity and makes errors by inserting the incorrect nucleotide more frequently than the wild-type enzyme.In addition to M19, several other T4 polymerase amber and temperature-sensitive mutants have been located on a linear, fine-scale map. The mutants which most strongly affect mutation rates are found in two clusters at 25% and 80% of the gene. These two domains may represent the active site(s) of the polymerase and exonuclease activities.