Multiple forms of DNA-ase activity in the nuclei of spleen lymphocytes]

The nuclei of spleen lymphocytes showed nuclease activity becoming manifest under conditions optimal for different types of DNA-ase (DNA-ase I, DNA-ase II, micrococcal nuclease and Ca, Mg-dependent endonuclease). No diversity of nuclease activity was found in the liver or kidney nuclei. A high nuclease activity in the lymphocyte nuclei provides for a deeper endonucleolysis of the lymphocyte chromatin as compared to that in the liver nuclei. The variety of nucleic activity and more advanced chromatin endonucleolysis in the spleen lymphocyte nuclei may be associated with rapid cell renewal of the lymphocyte pool in lymphoid organs and with necessity for autolysis of degraded lymphocyte genome. It may also ensure the somatomutagenic mechanism of diverse V-genes and of V- and C-gene combination.     

Dynamics of the endo-DNAase activity of cell nuclei of mouse thymus and spleen lymphocytes during the immune response

Endo-DNAse (mostly Ca/Mg-dependent endonuclease) activity was studied in extracts of lymphocyte cellular nuclei from the spleen and thymus of mice upon their immunization with sheep red blood cells. Endo-DNAses were detected by their action on super-stranded DNA pBR 322. It has been established that endo-DNAse activity considerably changes in the course of immune response. The changes start in the early (induction) phase of immune response, are characterized by certain regularities and are distinct in thymus and spleen lymphocytes. It is assumed that endo-DNAses of lymphocyte cellular nuclei are involved in antigen-dependent lymphocyte differentiation.     

Isolation of Ca/Mg-dependent endonuclease of cell nuclei as the basic component in the chromatin nuclease complex of human lymphocytes

Highly purified preparations of Ca/Mg-dependent cell nuclear endonuclease have been obtained from human spleen lymphocytes. The purification was 2000-fold, and the enzyme was a polypeptide with a molecular weight of 57-54 kD. The study of its binding with monoclonal antibody enzyme, produced by various hybridoma strains obtained upon the immunization of mice with non-fractionated nuclear extract has shown that Ca/Mg-dependent endonuclease was the best expressed endonuclease of lymphocyte cellular nuclei. Possible production of some other cell nuclear endonucleases during processing of CH/Mg-dependent endonuclease is suggested.     

Characteristics of the effect of Ca/Mg-dependent endonuclease from cell nuclei of human lymphocytes on J kappa-cluster of immunoglobulin genes

The cleavage of the murine J kappa-segments by purified Ca/Mg-dependent endonuclease was studied. The enzyme was shown to cleave specifically the J kappa-segments introducing the double strand breaks in 5'-regions of the J kappa-genes. The number of thus generated discreet fragments does not change during incubation. Specificity of the enzyme is not changed by substitution of Ca/Mg for Mn. The enzyme activity is inhibited by 0.1 M NaCl as well as its specificity. Comparison of the enzyme with the known nuclease activities that cleave specifically the J kappa-segments demonstrated that the activities of the latter are determined by Ca/Mg-dependent endonuclease. The results suggest the possible participation of Ca/Mg-dependent endonuclease in Ig-genes recombination.     

Ca/Mg-dependent endonuclease as a probe for detecting chromatin changes in lymphocytes in chronic lymphoid leukemia

Chromatin fragmentation of bovine peripheral blood lymphocytes from normal animals and the ones suffering from chronic lympholeucosis (CLL) by DNase I, micrococcal nuclease and purified Ca/Mg-dependent endonuclease from nuclei of human splenocytes was studied. The lymphocytes chromatin from CLL animals was shown to be more resistant to nucleases, than the one from normal animals. It was found that difference between fragmentation of chromatin samples from normal and CLL bovines was more dramatic when Ca/Mg- dependent endonuclease was used versus traditionally exploited DNase I and micrococcal nuclease. The data suggest that purified Ca/Mg-dependent endonuclease can be a useful enzymatic probe for detection of lymphocytes chromatin changes during CLL.     

Nuclear topoisomerase I and DNase activities in rat diethylnitrosamine-induced hepatoma, in regenerating and fetal liver.

DNase activity in the presence of Ca2+ + Mg2+, Mg2+ alone, Mn2+ alone, or EDTA, and topoisomerase I activity were measured in nuclear extracts of diethylnitrosamine (DEN)-induced hepatomas, regenerating, fetal, and normal rat livers. In hepatoma tissue, the Ca/Mg-dependent DNase activity was lower than in normal tissue and nearly the same as in fetal liver. In the poorly differentiated hepatomas, Mn-dependent DNase activity was higher than in both moderately and well differentiated ones and than in normal liver tissue. The activity of topoisomerase I in hepatomas and in regenerating liver was lower than in normal liver tissue.     

Isolation and analysis of Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes

A scheme for the isolation of Ca,Mg-dependent endonuclease from human spleen lymphocyte nuclei has been developed. The isolation procedure resulted in protein preparations (Mr = 57 kD) possessing an enzymatic activity and stable upon storage for over a period of one year. The enzyme is an endonuclease which predominantly cleaves double-stranded DNA by a mixed single- and double-hit mechanism with the formation of 5'-phosphate and 3'-OH terminal groups. Its maximal activation is induced by Ca2+ plus Mg2+. The enzyme is also active in the presence of Mn2+, Ca2+, Mg2+ and Zn2+ and is inhibited by Co2+. NaCl and KCl (0.15-0.2 M) and p-chloromercuribenzoate (1 mM) also inhibit the enzyme. ATP has no activating effect.     

hsBAFF对小鼠脾脏B淋巴细胞增殖与免疫应答及胞内Ca2+水平的影响

目的：探讨大肠杆菌表达的人源可溶性B淋巴细胞激活因子hsBAFF对小鼠脾脏B淋巴细胞免疫反应及其对胞内游离Ca^2＋信号变化的影响。方法：选择20只健康ICR小鼠,雌雄各半,随机分成两组（n=10）：①对照组;②hsBAFF实验组。实验组小鼠腹腔注射含hsBAFF（0．1mg／kgbw）的PBS溶液,对照组注射同等剂量的PBS溶液．连续8d。用MTT法检测小鼠脾脏B淋巴细胞的增殖及其对LPS刺激的免疫反应,并用激光共聚焦显微镜分析脾脏B淋巴细胞胞内钙离子水平（[Ca^2＋]i）变化。结果：hsBAFF注射小鼠的脾脏B淋巴细胞增殖和对LPS刺激的免疫反应均明显高于对照组（P〈0．05）;hsBAFF注射组[Ca^2＋]i荧光强度维持在相对稳定的高水平上波动,平均荧光强度显著高于对照组（P〈0．01）,且荧光强度变化率小于对照组。结论：大肠杆菌表达的hsBAFF能促进B淋巴细胞的增殖和免疫应答,从而增强机体免疫功能。hsBAFF激活小鼠脾脏B淋巴细胞可能与[Ca^2＋]i升高有关。     

Insulin-induced increases in the activity of the spontaneously active and ATP.Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver

Liver supernatant from normal and alloxan-diabetic rats was fractionated by DEAE-cellulose chromatography and the separated phosphoprotein phosphatase fractions were assayed with [32P]histone f2b, [32P]phosphorylase a and [32P]phosphorylase kinase as substrates. In diabetic rat liver, one of the phosphatase fractions found in the normal liver was significantly reduced. This fraction was identified as a mixture of the spontaneously active form and the ATP . Mg-dependent form of phosphoprotein phosphatase-1 (Fc) based on sensitivity to inhibitor-2, substrate specificity, and the fact that it could be activated 42-70% by glycogen synthase kinase-3 in the presence of ATP . Mg. Further analysis of this fraction showed that liver cytosol from diabetic rats contained 62-79% lower spontaneously active phosphatase-1 activity and 40-51% lower combined spontaneously active and ATP . Mg-dependent protein phosphatase-1 (Fc) activity. Insulin administration increased the spontaneously active and the ATP . Mg-dependent protein phosphatase-1 activities approximately 45% and 36%, respectively, in alloxan-diabetic rats. These data imply that the lower levels of spontaneously active phosphatase-1 activity in diabetic rat liver cannot be explained by presuming phosphatase-1 to have been present as Fc, the inactive form. Moreover, insulin restored the total activity of the spontaneously active and activatable forms of phosphatase-1 to those present in normal liver implying that both forms of phosphatase-1 activity are under hormonal control.     

Circulating immune complexes as possible cause for anticomplementary activity in humans with malignant melanoma

Summary Sera from 98 melanoma patients, 20 noncancer patients with immune complex-associated diseases, and 90 normal donors were analyzed for anticomplementary (AC) activity by the complement consumption method. Some of these sera were also tested for immune complex-like materials by the Raji cell radioimmune assay. In addition, serum samples from ten melanoma patients were analyzed serially to correlate the AC activity with clinical course. Significant levels of Ac activity were found in 45% of melanoma sera, 75% of nonmalignant immune complex-associated disease sera, and 10% of normal donors' sera. In some patients, AC activity decreased and became undetectable as their disease progressed. AC-negative serum samples taken from melanoma patients late in the course of disease when the tumor burden was large became anticomplementary when mixed with autologous or allogeneic serum samples taken earlier at the time of little or no tumor burden. The early serum samples contained antibodies against autologous tumor extracts, as shown by a complement fixation test. Absorption of early serum samples with cultured allogeneic melanoma cells reduced their ability to consume complement when mixed with autologous late serum samples, suggesting the presence of free antigen in the latter. The mixed samples of early and late sera and the sera positive in the complement consumption test contained heavy nonmonomeric IgG. Therefore, the AC activity of melanoma sera could be due to tumor-associated antigen-antibody complexes.     

On the role of Ca, Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues

The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.     

Peculiarities of metabolism of UV-irradiated lymphocytes

The influence of UV-light (240-390 nm) at a dose of 151 and 755 J/m2 on the functional properties of lymphocyte metabolism key enzymes from donors' human blood: lactate dehydrogenase (LDH), cytochrome c oxidase, succinate dehydrogenase (SDH), Ca2(+)-ATPase of plasma membranes has been investigated. It has been revealed that photoinactivation of enzymes immediately after UV-irradiation which leads to the decrease of the ATP content in lymphocytes is replaced by the increased activity of the enzymes under investigation during daily incubation of lymphocytes. As a result, the level of ATP in photo-modified lymphocytes does not differ from that in native cells before incubation. This indicates the normalization of biochemical processes in lymphocytes influenced by UV-light applied in autotransfusion of UV-irradiated blood.     

Alteration of signal-transducing molecules in tumor-infiltrating lymphocytes and peripheral blood T lymphocytes from human colorectal carcinoma patients

 Tumor development or growth is accompanied by impaired immune responses, such as a poor proliferative response or down-regulated cytolytic T lymphocyte activity. Although recent reports have suggested that modification of the signal-transducing molecule is responsible for impaired immune responses in tumor-bearing hosts, the causes of defective immune function are not yet completely understood. Furthermore, the clinical significance of the findings is not yet clear. In this study, we investigated the alteration of several signal-transducing molecules in peripheral blood T lymphocytes (T-PBL) as well as in tumor-infiltrating lymphocytes (TIL) from human colorectal carcinoma patients and their relationship with the impaired host immune responses. A greater reduction in CD3ζ chain level was observed in TIL than in T-PBL from tumor-bearing hosts. CD3ζ chain reduction in T-PBL correlated with the clinicopathological stage of a tumor, especially with the status of lymph node metastasis. The levels of p56 ^lck and p59 ^fyn protein tyrosine kinase in T-PBL were also compared between tumor-bearing hosts and normal healthy volunteers. In T-PBL from tumor-bearing hosts, expression of protein tyrosine kinase p59 ^fyn was significantly lower than that of p56 ^lck . However, the level of CD3ζ chain expression did not correlate with T lymphocyte functions such as T lymphocyte proliferative response or allogeneic target cell lysis. Received: 25 September 1996 / Accepted: 25 August 1997     

Dysfunction of protein kinase FA/GSK-3α in lymphocytes of patients with schizophrenic disorder

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP. Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA /GSK-3α immunoblotting and kinase activity analysis in an anti-kinase FA /GSK-3α immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA /GSK-3α in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA /GSK-3α activity in the high levels of 14.8 ± 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA /GSK-3α activity in the low levels of 2.8 ± 1.6 units/mg, indicating that the different levels of kinase FA /GSK-3α activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide intial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA /GSK-3α, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes. © 1995 Wiley-Liss, Inc.     

痰湿体质PCOS临床特征及T淋巴细胞亚群分布特征分析

目的:探讨痰湿体质多囊卵巢综合征患者临床特征及T淋巴细胞亚群分布特征。方法:根据PCOS鹿特丹诊断标准和王琦教授痰湿体质的判定标准,选择痰湿体质PCOS患者、非痰湿体质PCOS患者各30例,正常对照组15例进行性激素和糖脂代谢指标检测和T淋巴细胞亚群检测,明确痰湿体质PCOS患者临床特征及T淋巴细胞亚群的分布特征。结果:三组在年龄和体重指数上无差异,具有可比性;痰湿体质PCOS与非痰湿体质PCOS在LH、T、30 min INS、120 min INS、CHO、LDL水平明显高于对照组,具有统计学意义(P0.05);痰湿体质组OGTT血糖水平、CHO、LDL、IR水平明显高于非痰湿体质组,差异具有统计学意义(P0.05)。痰湿体质、非痰湿体质PCOS患者外周血CD4+T淋巴细胞、CD4+/CD8+明显高于对照组,且痰湿体质PCOS患者CD4+T淋巴细胞、CD4+/CD8+明显高于非痰湿体质PCOS患者,差异有显著性(P0.05)。结论:PCOS患者存在糖脂代谢紊乱,其中痰湿体质患者较非痰湿体质患者更明显;PCOS患者存在细胞免疫异常,痰湿体质患者较非痰湿体质患者更易出现T淋巴细胞亚群异常,说明痰湿体质PCOS的发生与免疫因素关系更密切。     

The effect of irradiation and alkylating agents on chromatin breakdown in normal and malignant lymphoid cells

Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chromatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/BL cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes.     

Role of the modulator protein in the interconversion of rabbit skeletal muscle protein phosphatase

The major active protein phosphatase present in a rabbit skeletal muscle extract is associated with the glycogen particle and migrates in sucrose density gradient centrifugation as a Mr = 70,000 protein and contains modulator activity. Addition of extra modulator protein causes a time- and concentration-dependent conversion of the enzyme to an inactive FA-ATP, Mg-dependent form. The intrinsic modulator in the active phosphatase is destroyed by limited proteolysis without an appreciable change in the phosphatase activity. The proteolyzed active enzyme has a lower molecular weight (Mr = 40,000) and it reassociates with the modulator producing a FA-ATP, Mg-dependent enzyme form (Mr = 60,000). The modulator protein is used stoichiometrically in the activation of the ATP, Mg-dependent phosphatase. This is in agreement with the presence of one unit of modulator activity per unit of native spontaneously active phosphatase.     

Specificity of fragmentation of DNA from pBR322 plasmid by Ca,Mg-dependent endonuclease from cell nuclei of human lymphocytes

Fragmentation of the plasmid pBR322 DNA by a purified preparation of Ca/Mg-dependent endonuclease has been studied. It was shown that on the first steps of reaction the double-stranded cuts are introduced into the superhelical DNA independent of singlestranded ones. The doublestranded cuts are introduced into superhelical and linear DNA in 12 sites enriched with GC-pairs, 9 of them include pentanucleotide CGCGG(CCGCC) that is functionally significant. Relaxation of the plasmid DNA by topoisomerase I blocks the sitespecific action of the enzyme. Ca/Mg-dependent endonuclease is concluded to be topologically dependent enzyme, possibly, participating in the recombination processes.     

Inhibition of mitogen-induced lymphocyte proliferation correlated to anticomplementary activity in sera from melanoma patients

Summary Sera from 98 melanoma patients and 90 normal donors were analyzed for antibody (Ab) to melanoma extracts, melanoma-associated antigen (Ag) and anticomplementary (Ac) activities by the microcomplement consumption technique. Sera were also tested for their ability to inhibit mitogen(phytohemagglutinin: PHA)-ininduced blastogenesis of normal lymphocytes. The results of the complement consumption assays were correlated with the results of inhibition of PHA-induced blastogenesis. Of 98 melanoma sera, 22% were Ab-positive, 30% were Ag-positive, and 44% were Ac-positive, in contrast to only 6% Ab-positive, no Ag-positive, and 7% Ac-positive in 90 normal sera. Fifty-nine percent of melanoma sera were inhibitory to PHA-induced blastogenesis, as against 12% of normal donors' sera. Ac-positive melanoma sera were significantly more inhibitory than Acnegative melanoma sera. The inhibitory activity of Acpositive sera was potentiated by the simultaneous presence of detectable Ag activity and was diminished by detectable Ab activity. Presence of Ab or Ag activity alone did not correlate with the inhibitory activity of the melanoma sera. Increasing incidence of Ac activity and ability to inhibit PHA-induced blastogenesis was observed with increasing tumor burden or advancing clinical stage. Sera from patients who displayed a delayed cutaneous hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) were less inhibitory to PHA-induced blastogenesis than the sera from patients who did not respond to this contact allergen. Thus, Ac activity may be one of the circulating factors responsible for the immunosuppressive effect of cancer patients' sera.     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.