Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

The immunomodulating properties of staphylotoxin]

The action of native staphylotoxin has been tested on T lymphocytes obtained from 21 healthy human donors. As revealed in this investigation, toxic action affects Fc-receptors to IgG, IgM and IgA. The dose-dependent effect of the preparation suggests its direct contacts with Fc-receptors of lymphocytic membranes. High doses of the toxin suppress the expression of Fc-receptors 6- to 12-fold, low doses of the toxin are capable of stimulating the expression of Fc gamma-receptors.     

Fragmentation of DNA lymphocytes the human in dynamics of development apoptosis, induced influence of UV-radiation and reactive oxygen species

It is established that UV-light (240-390 nm) in doses of 151, 1510 and 3020 J/m2 and reactive oxygen species and singlet oxygen induce DNA fragmentation lymphocytes cells of the human 20 h after influence. Using a method of DNA-comets it is revealed that DNA damages (single strand breaks) are found out right after UV-irradiations of lymphocytes in doses of 1510 and 3020 J/m2 and additions hydrogen peroxide in concentration of 10-6 mol/l (a comet of type C1) and reach a maximum through 6 h after influence on of cells UV-light and ROS (comets of types C2 and C3). Assumption about the leading part of a p53-dependent way in realization apoptosis human lymphocytes in the conditions of influence of UV-light and reactive oxygen species is put forward.     

Effect of ionizing radiation on Fc-receptors of mouse peritoneal mononuclear cells

The influence of different gamma-radiation doses on the number of mouse peritoneal mononuclears bearing Fc-receptors was studied. At doses of 7 and 9.5 Gy, phase changes were observed in the number of cells bearing Fc-receptors: a short-term rise registered 24 h to 2 days after irradiation was followed by depression. In addition, the total number of peritoneal mononuclears decreased. At a dose of 100 Gy, the number of cells with Fc-receptors was reduced as early as 1 h after irradiation, and the pronounced cell lysis occurred.     

Change in spectral properties of nitrosohemoglobin induced by UV light

The effect of UV radiation on the spectral characteristics of human nitrosohemoglobin in a wide dose range (151-4530 J/m2) was studied. It was shown that the irradiation of hemoprotein solutions with low doses of UV light (151-453 J/m2) led to local conformational rearrangements of the iron porphyrin moiety of the molecule. The apoprotein exhibited a high photostability under the conditions of the experiment. High doses of UV radiation (1359-4530 J/m2) induced the accumulation of methemoglobin in the protein sample. A scheme of the photoinduced formation of methemoglobin was elaborated.     

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.     

Apoptosis and necrosis induced by ultraviolet radiation in the presence of autologous plasma

This paper deals with the effect of ultraviolet (UV) light (240–390 nm) at doses of 151–3020 J/m²* on the character of the death of donors’ blood lymphocytic cells (with the use of markers of apoptotic and necrotic cell death) and on the level of the CD95 receptor—a marker of apoptotic predisposition. UV irradiation has been shown to increase the expression of CD95 receptors, which is due mainly to synthesis de novo. It is revealed that over the course of the 24-h incubation of photomodified lymphocytes (at irradiation doses of 151 and 755 J/m²) without autologous blood plasma, cell death occurs by receptor-mediated apoptosis. Action of high irradiation doses (1510 and 3020 J/m²) leads to the massive necrotic death of immunocytes. The use of autologous blood plasma during the incubation of photomodified lymphocytes allows a reduction in the amount of both apoptotic and necrotic cells.     

Covalent-bound aggregates of equine venezuelan encephalomyelitis virus induced by UV-irradiation

Formation of Venecuelan equine encephalomyelitis virus (VEE) aggregates induced by UV-light has been studied. The high doses of UV-irradiation induced the protein-protein cross-links resulting in formation of fast sedimenting viral structures. The latter structures are supposed to be presented by the aggregates of several virions linked by the UV-light induced RNA-protein and protein-protein covalent bonds. The lesions in the fine structure of virion envelope was registered by the electron microscopy technique.     

The 3H-thymidine incorporation into G0 human lymphocytes induced by low doses of UVC radiation

The 3H-thymidine incorporation in human lymphocytes of healthy donors induced by UVC radiation under doses 0.0008-60 J/m2 was investigated. It was shown that the incorporation of 3H-thymidine increases under doses in interval 0.1-20 and is constant under doses higher than 20 J/m2. Under doses in interval 0.006-0.03 J/m2 near a half of all samples had the level of incorporation increased in comparison with control samples. We connect the presence, absence or variability of this effect with individual peculiarities of cells and with different activity of cell subpopulations that are different on morphological and physiological characteristics. The hypothesis about the role of this factor in the influence of low doses of pathogenic agents (UVC and X-radiation, chemical compounds) on human lymphocytes is discussed.     

Level of receptor complex molecule expression of human blood T-lymphocytes under conditions of their UV-irradiation

The influence of a wide range of doses of UV-light (240-390 nm) on the expression level of receptor complex molecules (CD3, CD4, CD8 markers) on the membrane surface of human blood T-lymphocytes has been studied using flow cytometry and enzyme linked immunosorbent assay. UV-light at small and medium doses (15 1, 453 and 906 J/m2) has been established to have a unidirectional (activating) effect on the expression level of receptor complex molecules, and at a high dose of 1359 J/m2 it can either increase (CD4 and CD8 markers) or reduce (CD3 complexes) the quantity of the analyzed molecules on the membrane surface of T-lymphocytes.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Indicators of cellular and humoral immunity in chronic opisthorchiasis]

Quantitative content of T- and B-lymphocytes in the peripheral blood was studied after Mendes in 30 patients suffering from chronic opisthorcosis and in 30 practically healthy persons; immunoglobulins--M, G, and A were examined after Mancini, and the presence of autoantibodies in the blood serum was studied by indirect method of mast cells degranulation. There was revealed a reduction of the T-cell count to 28.8 +/- 2.28% (normal value--52.9 +/- 3.32%) and a fall of IgM level to 163.4 +/- 16.2 (207.2 +/- 10.5 in donors) in the patients; macrophages count was almost doubled. Autoantibodies to the antigens of the liver, gastric mucosa and the gall bladder wall were revealed in 1/3 of the patients.     

The Humidity Response of Stomata and its Measurement

Meidner, H. 1987. The humidity response of stomata and its measurement.—J.exp. Bot. 38:877–882. Using artificial membranes of constant conductance with vapourpressures below the membranes of 2?33 kPa (saturated) and 2?26kPa (97% saturated) it was found that an automated water vapourdiffusion porometer registered a change in conductance. In otherwords, it underestimated the conductance when the vapour pressurebelow the membranes was only 97% saturated. This is becausethe programmed computation of the conductance assumes that thespace below the membrane is at all times saturated. The implicationsfor measuring leaf conductance with these instruments is discussedin connection with the humidity response of stomata to a stepwiseincrease in the vapour pressure difference between the leafinterior and the atmosphere. This response appears to involvea lowering of the vapour pressure in the sub-stomatal spaceand, therefore, a transient opening movement which can be detectedby sensitive mass flow porometry. It is concluded that in cases in which a transient opening movementprecedes partial stomatal closure the humidity response to anincrease in vapour pressure difference cannot be due to vapourloss direct from the guard cells. Key words: Humidity, transients, stomata     

Production and study of the properties of a polyvalent corpuscular Pseudomonas aeruginosa vaccine. IV. The effect of immunization with Pseudomonas aeruginosa vaccine on the immune status of volunteer donors

The influence of immunization with P. aeruginosa vaccine on the immune status of volunteer donors has been studied. Immunization with P. aeruginosa vaccine in doses of 0.5-0.5-1.0 ml at intervals of 7 days has been found to lead to the 13-fold increase of the titer of specific antibodies, lasting for 3-4 months, which ensures the possibility of obtaining anti-P. aeruginosa hyperimmune plasma with standard titers. The injection of P. aeruginosa vaccine to donors leads to the activation of humoral immunity simultaneously with the increase of the absolute and relative number of rosette-forming B-lymphocytes without essential changes in the amount of rosette-forming T-lymphocytes.     

Transient activity of Golgi-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro

Previous reports demonstrated that the vesicular stomatitis viral glycoprotein (G protein), initially present in membranes of a Chinese hamster ovary mutant cell line (clone 15B) that is incapable of terminal glycosylation, can be transferred in vitro to exogenous Golgi membranes and there glycosylated (E. Fries and J. E. Rothman, 1980, Proc. Natl. Acad. Sci. U. S. A. 77:3870-3874; and J. E. Rothman and E. Fries, 1981, J. Cell Biol. 89:162-168). Here we present evidence that Golgi-like membranes serve as donors of G protein in this process. Pulse-chase experiments revealed that the donor activity of membranes is greatest at approximately 10 min of chase, a time when G protein has been shown to have arrived in Golgi stacks (J. E. Bergmann, K. T. Tokuyasu, and S. J. Singer, 1981, Proc. Natl. Acad. Sci. U. S. A. 78:1746-1750). Additional evidence that the G protein that is transferred to exogenous Golgi membranes in vitro had already entered the Golgi membranes in vivo was provided by observations that its oligosaccharides had already been trimmed, and that its distribution in a sucrose density gradient was coincident with that of enzymatic markers of Golgi membranes. The capacity of this Golgi-like membrane to serve as donor is transient, declining within 5 min after "trimming" in vivo as the G protein enters a "nontransferable" pool. The rapidity of the process suggests that both the "transferable" and "nontransferable" pools of G protein reside in Golgi-like membranes.     

Inactivation of single-celled Ascaris suum eggs by low-pressure UV radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m2 for intact eggs and from 0 to 500 J/m2 for decorticated eggs. With a UV fluence of 500 J/m2, 0.44-+/-0.20-log inactivation (mean+/-95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m2 resulted in 2.23-+/-0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m2), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m2), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80-+/-0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m2, suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-microm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (approximately 20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

Fc receptors and surface immunoglobulins in cells of hairy cell leukemia]

Using 125I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')2-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It was found that all cells containing this enzyme bore sigma-chains on their surface. On more than 90% of these cells a simultaneous expression of mu-chains was detected. gamma-Chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.     

Protein phosphorylation regulated by cyclic nucleotide-dependent protein kinases in cell extracts and in intact human lymphocytes.

A specific 46,000/50,000 molecular weight protein substrate for both cAMP-dependent protein kinase (cAK) and cGMP-dependent protein kinase (cGK) extensively characterized and purified from human platelets was found to be present also in human T-lymphocytes, B-lymphocytes and other cells and tumour cell lines. This protein termed vasodilator-stimulated phosphoprotein (VASP) was present in cytosol and membranes of lymphocytes. Addition of exogenous purified cAK or cGK to lymphocyte cytosol or membranes converted 80-90% of VASP to its phosphoform. Endogenous VASP phosphorylation in both cytosol and membranes was stimulated by the addition of cAMP but not by cGMP. With intact lymphocytes, prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) induced an increase of cAMP and converted 70% of VASP to its phosphoform. In contrast, an increase of cGMP was not associated with VASP phosphorylation although cGK was detected in lymphocytes. These data support the hypothesis that VASP phosphorylation may be an important component of cAMP-mediated regulation of lymphocyte function.     

Distribution of HLA antigens in the armenian population of Nagornyi Karabakh

Characteristics of the distribution of 31 HLA antigens of classes I (A, B, and Cw) and II (DR) in Nagornyi Karabakh Armenians are reported for the first time. It has been found that the antigens most common in this population are A2, A3, A9, B5, B7, B12, Cw4, DR4, DR2, and DR3; the least common antigens are B15, B16, and B40. The results are compared with the data for Armenians living in Armenia and those for major ethnic groups. The frequencies of HLA antigens in Nagornyi Karabakh Armenians match those in Armenians living in Armenia. In the HLA-antigen distribution, Armenians are generally close to Caucasoids.