Chromosomal aberrations induced in human cultured cells by liposome-encapsulated deoxyribonuclease I

Experiments of incorporation of a nucleolytic enzyme into human cells cultured in vitro have been carried out with the aim of inducing structural chromosome variations. Human heteroploid cells, either as asynchronous populations or enriched in mitoses, and PHA-stimulated lymphocytes were used as recipients. We found that all these cells when exposed to pancreatic DNAase I encapsulated in liposomes, either of multilamellar (MLV) or of small unilamellar (SUV) type, show an incidence of chromosome damage higher than that induced by the enzyme free in the incubation buffer. Our results indicate that liposomes are suitable vehicles for the transfer of an exogenous nuclease into human cultured cells. The enzyme remains functionally active and interacts with nuclear DNA, giving rise to chromosome lesions.     

Non random distribution of lesions induced by deoxyribonuclease I in human chromosomes

We studied the action of deoxyribonuclease I on human lymphocytes in order to determine the localization of the DNAase induced aberrations. Our results indicate a non-random distribution of the lesions on chromosome regions which may reflect a differential pattern of sensitivity to the enzyme. Furthermore we observed a correspondence between the preferential DNAase induced breaks and fragile sites that are expressed in lymphocytes maintained in medium without folic acid. A possible interpretation of our findings is that the accessibility to DNAase and/or the efficiency of the repair systems depend on the chromatin structure that influences also the expression of some common fragile sites.Abbreviations DNAase I deoxyribonuclease I E.C.3.1.4.5.     

Multiple forms of deoxyribonuclease I

Summary This article will review recent progress on the purification of DNase I (E.C.3.1.4.5) from various sources and the characterization of multiple forms of the enzyme. The chemical basis of the multiple forms in bovine pancreas will be discussed in detail, while for other DNases, including those in ovine pancreas, bovine, mouse and rat parotid, and malt, only the evidence for multiplicity will be presented.     

Studies on dilution inactivation of sheep liver pyruvate carboxylase

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.     

重组肝素酶I热失活动力学模型的建立与应用

对组氨酸标签肝素酶I（His．HepI,Ec4．2．2．7）的热失活机制进行了研究。针对短暂低温处理可以使热失活His．HepI酶活部分恢复及添加二硫苏糖醇（D1Tr）使其热稳定性提高的现象,利用荧光探针法研究了失活过程His．HepI构象变化,证明了该酶构象存在可逆转变行为。为进一步明晰His．HepI的热失活机制,假设该酶热失活的主要途径包括去折叠及形成聚集体,并以此为基础建立模型进行拟合,模型与实验值吻合良好,表明假设的合理性。根据模型计算的活化能为Er=100．217kJ／mol、Eir=7．857kJ／mol和Ed=77．062kJ／mol,此数据从一定程度上解释了冷处理为何能使His．HepI部分恢复活性。进一步研究表明,任何能够抑制这两种途径发生的措施对于提高His．HepI热稳定性都是有效的。     

Effect of antigenic stimulation on acid deoxyribonuclease activity in lymphocytes: I. Antibody-induced response

The effect of antigen-induced stimulation on acid deoxyribonuclease (DNase) activity in BALB/c mouse lymphoid cells was determined. Increase in acid DNase activity was found in intact spleen cell populations of mice from the second or fourth day after immunization with pneumococcal polysaccharide type III and from the fourth day after immunization with SRBC. DNase determinations performed with spleen cell fractions prepared from SRBC-immunized mice, showed that the rise in the enzyme activity was confined to the fraction containing the antibody-forming cells. The DNase activity was also increased in spleen cell cultures, stimulated with SRBC in vitro. Rise in the activity of this enzyme was also observed in peritoneal cell populations taken from SRBC-immunized mice. This change was maximal on the second day after immunization, when no appreciable increase in DNase activity of spleen cells was yet detected. The results obtained suggest, that acid DNase is an enzyme involved in the proliferative/maturation response to antigenic stimulation. It is a consequence of antigenic stimulation rather than being involved in the process of afferent stimulation.     

Depolymerization of F-actin by deoxyribonuclease I.

Deoxyribonuclease I causes depolymerization of filamentous muscle actin to form a stable complex of 1 mole DNAase I:1 mole actin. The regulatory proteins tropomyosin and troponin bind to filamentous actin and slow down but do not prevent the depolymerization. In the absense of ATP, heavy meromyosin binds tightly to actin filaments and blocks completely the DNAase I: actin filament interaction. Addition of ATP releases heavy meromyosin; DNAase I is then rapidly inhibited and the actin filaments are depolymerized.     

Genetic polymorphism of human urine deoxyribonuclease I

Summary A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes —DNASE1 1, 1–2, and 2 — and the other four rare phenotypes — DNASE1 1–3, 2–3, 2–4, and 3–4 — represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 ^* 1, ^* 2, ^* 3, and ^* 4. The frequencies of the DNASE1 ^* 1, DNASE1 ^* 2, DNASE1 ^* 3, and DNASE1 ^* 4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.     

Effect of ionic liquids alkyl chain length on horseradish peroxidase thermal inactivation kinetics and activity recovery after inactivation

The effects of different alkyl chain lengths of ionic liquids 1-ethyl-, 1-butyl- and 1-hexyl-3-methylimidazolium chloride, on the catalytic activity, thermal stability and deactivation kinetics of horseradish peroxidase were studied in the temperature range of 45–85 °C. The presence of 1-ethyl- and 1-butyl-ionic liquids up to 25 % (w/v) did not affect significantly the enzyme activity at 25 °C, whereas the addition of 1-hexyl-solvent resulted in lower activity of enzyme. Typical biphasic deactivation profiles were obtained and adequately fitted by a bi-exponential equation. When increasing ionic liquids concentration up to 25 % (w/v), the second phase of deactivation became more prominent, till leading to apparent first-order kinetics. Occurrence of activity regain, following thermal deactivation was found, reaching up 60–80 % of the initial activity, especially in 1-hexyl-3-methylimidazolium chloride. Activity regain was particularly noticeable in the first phase of deactivation. Temperature sensitivity of the Soret band maxima indicated that the enzyme prepared in buffer or 1-hexyl-3-methylimidazolium chloride had similar conformational changes in the haem region, but no correlations were found with activity decrease.