Changes in nucleotide DNA structure and adhesive properties of blood leukocytes in early period after irradiation of animals]

Postirradiation changes of adhesive properties and supercoiled nucleoid DNA structure of blood leukocytes were studied in Macaca nemestrina and in rats. The dynamics of membrane changes demonstrated a temporary increase of the leukocyte adherence 24 h after nonlethal irradiation of rats, followed by a decrease of this parameter to normal levels on h 48. After lethal irradiation of both animal species the increase in adhesive leukocytes fraction was shown already on hr 3. This higher index was observed until the end of experiments (5 days). Early (on h 3-6) temporary loosing of supercoiled DNA structure was demonstrated in the total blood leukocytes of nonlethally irradiated animals, this seems to be connected with lymphocyte changes. This process was not dependent on altered adhesive properties of leukocyte membranes. The membrane changes of leukocytes preceded decondensation of supercoiled DNA after lethal irradiation of animals. Loosing of supercoiled DNA progressively increased on hour 24 and in later postirradiation period.     

Vitamin E, ubiquinone and ubiquinone-dependent enzymes in experimental myocarditis

It is shown that a day after introduction of adrenaline which evokes experimental focal myocarditis the level of ubiquinone and vitamin E content in the myocardial mitochondria increases by 56.8 and 122%, respectively. Succinate-ubiquinone-reductase activity in mitochondria remains practically unchanged, while NADH-ubiquinone-reductase activity considerably falls. 5 days after the focal myocarditis reproduction the content of ubiquinone and NADH-ubiquinone-reductase activity of mitochondria return to the norm, while the vitamin E amount remains higher than in intact animals. 24h after adrenaline introduction the level of succinate-ubiquinone-reductase activity of blood leucocytes considerably grows. It is not normalized even on the 5th day after adrenaline administration. It is supposed that the level of this activity may be an index showing development of the focal myocarditis.     

Effects of Injury on the Indoleamines in Cerebral Cortex

It was shown previously that focal cortical freezing lesions in rats cause widespread depression of local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere. This was interpreted as reflecting functional depression. The underlying mechanisms were postulated to involve alterations of biogenic amine systems. Accordingly, levels of serotonin (5-HT), its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and its precursor tryptophan were determined by an HPLC method with electrochemical detection in frontoparietal cortical areas of both hemispheres at 4 h and 1, 3, 6, 8, and 10 days after a unilateral cortical freezing lesion. The 5-HT content was significantly lower than normal in the lesioned hemisphere only at 24 h, whereas the 5-HIAA level peaked at 24 h but was significantly elevated above normal values between 4 h and 6 days after lesioning. No changes were noted in 5-HT and 5-HIAA contents in the hemisphere contralateral to the lesion. These results indicate that cortical 5-HT metabolism is increased throughout the lesioned hemisphere of a focally injured brain. The increase in tryptophan content of the lesioned brain appeared to have a time course more closely related to previously demonstrated changes in cortical LCGU than to the increase in 5-HIAA content.     

Stimulation of growth of serum-deprived chick embryo fibroblasts by 3',5'-phosphodiesterase.

In chick embryo fibroblasts (CEF) deprived of serum, DNA synthesis is reduced to a basal level in about 12 h, cell division ceases after 24–36 h and their morphology changes to a rounded, less refringent form. During several days without serum the cAMP content of the cells showed a slow increase or a maintenance of the level found before serum was removed. When CEF deprived of serum for 24 h were treated with beef heart 3′,5′-phosphodiesterase (PHD) the cAMP level fell about 40% after 3 h, ³H-thymidine incorporation into DNA was strongly stimulated with a peak of incorporation at 12 h after the start of PHD treatment, cell morphology returned to that observed before serum deprivation, and at 24 h there was an evident growth in cell population, with a parallel increase in protein content. The growth stimulation by PHD is transitory: after cells had been deprived of serum for 4 days the PHD effect was no longer noticeable on the above parameters. Theophylline (1 mM and 4 mM) inhibited the PHD-mediated stimulation of ³H-TdR incorporation, this could well have been due to its general toxic effect on the cells (see Discussion).     

The effect of hydrocortisone on neutrophil functional activity

The male (CBA X C57BL) FI mice received 125 mg of hydrocortisone per kg body weight intraperitoneally. The functional activity of neutrophils has been evaluated by means of nitroblue terazolium test (NBT-test) values taken before or after heat-killed S. marcescens cell stimulation in vitro by 2, 12, 24 h 3, 7 or 14 days post hormonal treatment. Throughout the 1st day after hydrocortisone administration the NBT-test values taken prior to as well as post microbial neutrophil stimulation were steadily increased. This effect could be seen as early as 2 h post hormone administration and it was linked with growing leukopenia and total decrease of blood granulocyte content. By the 3rd day the same very values turned up to become normal. The NBT-test values after microbial stimulation of neutrophils were 1.7 or 2.4 lower after hydrocortisone had been added to blood in vitro in a dose 3.5 X 10(-6) M or 7 X 10(-5) M.     

靛玉红治疗慢性粒细胞白血病作用原理的研究

 靛玉红是一种新型抗癌药物,治疗慢性粒细胞白血病(CML)有较好的疗效。本文通过体内及体外实验采用靛玉红—脂质体为剂型,观察药物对CML细胞的作用。据荧光偏振度的测定结果表明,此药物分子能降低大白鼠白细胞膜流动性。还测定服药3天的患者外周白细胞中DNA聚合酶Ⅰ的活性,比治疗前降低74±1％。为靛玉红抑制CML细胞DNA合成的机理提供依据。此外,还使用载有靛玉红的脂质体进行体外实验,表明靛玉红可以直接降低细胞膜的流动性以及抑制CML细胞中DNA聚合酶Ⅰ活性。对靛玉红治疗CML的作用机理提出一些看法和讨论。     

Comet assay to determine DNA damage induced by food deprivation in rats

The aim of this work was to evaluate, by comet assay, the possible inducing of DNA lesions in peripheral blood mononuclear cells of rats subjected to acute or chronic food deprivation. Wistar male rats were subjected to 72 h of partial (50%), or total acute food deprivation, and then allowed to recover for different time periods (24, 48 and 72 h). In other experiments, comet scores were determined in peripheral blood mononuclear cells of rats subjected to chronic food deprivation (25% and 50%) for 50 days. Blood aliquots were obtained before, during and after food deprivation. Comet assay was carried out, the comet units photographed and scored (class 0 up to 3). Acute and chronic food-deprived rats presented peripheral blood mononuclear cells with DNA lesions (comet classes 1, 2 and 3) and a significant increase (p<0.05) in the number of comet units compared with its basal level. The increase was proportional to acute food deprivation time, but after being taken off, it progressively returned to basal level after 48 h (partial group) or 72 h (total group). Chronic food-deprived rats presented a progressive increase of comet score up to 5 days, and a decrease thereafter to reach a basal level. Possible mechanisms of DNA lesions are discussed.     

Effect of putrescine on oxyntic gland and colonic mucosal growth in rats

The effect of putrescine on oxyntic gland and colonic mucosal growth was studied by measuring the rate of [3H]-thymidine incorporation into mucosal DNA in vitro (DNA synthesis) and DNA, RNA and protein content of the mucosa following intramuscular injections of the compound (50 mumoles/100g). Saline injected animals served as controls. Multiple injections of putrescine during a 2-day fast produced a significant enhancement of mucosal DNA synthesis in oxyntic gland and colonic mucosa, with no apparent change in DNA, RNA or protein content in either of the tissues, compared to the corresponding saline-controls, when measurements were made 12-24 h after the last injection. However, when the animals were killed after 4 days, DNA, RNA and protein content of oxyntic gland mucosa, and DNA and protein content of colonic mucosa were found to be significantly higher than in the respective saline-controls. We conclude that putrescine, taken up from the blood, can stimulate growth of gastrointestinal mucosa.     

Cytofluorimetric research on the changes in the DNA content in the nuclei of Amoeba proteus during prolonged starvation and after refeeding]

Dividing amoebae were manually selected from the culture of Amoeba proteus, and so groups of synchronously dividing (synchronized) amoebae were obtained. These synchronized amoebae were maintained without food. In spite of starvation, individual amoebae in some particular groups were seen to divide, whereas in other groups of amoebae there was no division at all. The starving amoebae died not earlier than 2 weeks after the last division. A relative DNA content in isolated nuclei has been determined cytofluorometrically for each of 6 groups of synchronized starving amoebae, unable to divide. The nuclei were isolated in different intervals after division (after the feeding was ceased): 1.0-1.5 h, 1 day and up to 13 days with 1-2 day intervals. In the all groups of amoebae DNA synthesis occurred on the first 1-2 days after division. The nuclear DNA content in amoebae of 3 groups increased more than two-fold as compared with the 1 h level, in other 3 groups the nuclear DNA content did not exceed the doubled 1 h level, but probably exceeded the doubled postmitotic level. Later on, the nuclear DNA content in starving amoebae of each group was seen to decrease by 16-20%. Amoebae of 3 of the 6 groups were given the food organisms (Tetrahymena pyriformis) 8 days after division (after cessation of feeding). 2-3 days after refeeding some of these amoebae divided, and the nuclear DNA content of the refed amoebae proved to be higher than that in amoebae that continued to starve. It is suggested that the decrease of DNA content in the nuclei of starving amoebae and the increase of DNA quantity in the nuclei of refed amoebae may result from degradation and induction of synthesis of specific extra DNA synthesized in amoeba nuclei during each cell cycle.     

Sequence-dependent DNA condensation and the electrostatic zipper

Sequence-dependent configuration changes and condensation of double-stranded poly(dG-dC).(dG-dC) (GC-DNA) and ds poly(dA-dT).(dA-dT) (AT-DNA) were observed by atomic force microscopy in the presence of Ni(II). Less condensing agent was required to generate configuration changes in GC-DNA as compared to AT-DNA. In the presence of Ni(II) cations, GC-DNA adopted a Z-type conformation and underwent a stepwise condensation, starting with partial intramolecular folding, followed by intermolecular condensation of two to several molecules and ending with the formation of toroids, rods, and jumbles. GC-DNA condensates were unusual in that the most highly condensed regions were surrounded by loops of ds GC-DNA. In contrast, AT-DNA retained its B-type conformation and displayed only minor condensation even at high Ni(II) concentrations. The Ni(II)-dependent differences in condensation between GC-DNA and AT-DNA are predicted by an extension of the electrostatic zipper motif proposed by Kornyshev and Leikin, in which we account for shorter than Debye screening length surface separations between the DNA molecules and for the Ni(II)-induced conformation change of GC-DNA to Z-DNA.     

Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs : A cytophotometric study

Mouse one-cell embryos were taken 1, 1.5, 2, 3, 4, 6, 8, 10, 13 and 18 h after insemination. One-cell parthenogenones were induced by treatment of mouse eggs obtained 20 h after HCG injection with hyaluronidase and cultured for 0.5, 1, 3, 4.5, 6, 8, 10, 12 and 24 h. Some parthenogenones were pulse-labelled with tritiated thymidine, cut and autoradiographed. Both the embryos and parthenogenones were Feulgen-stained, and integrated relative optical absorption of either pronuclei or nuclei of polar bodies was measured with a cytophotometer. In some fertilized eggs and parthenogenones the DNA synthesis sets in 4–6 h after either insemination or parthenogenetic stimulus. Between the 8th and 13th hour after insemination the fraction of DNA synthesizing embryonic pronuclei remained at the level 30–40%. Most parthenogenones duplicated their DNA content between the 8th and 12th hour after hyaluronidase treatment. The DNA synthesis time in pronuclei of embryos was determined to be 3.5–4.0 h and that of pronuclei of parthenogenones approx. 4 h. The minimal time of the G2 phase was estimated to be 3–5 h. The first labelled pronuclei of parthenogenones were detected 6 h after stimulus. Male pronuclei started and ended DNA synthesis earlier than female pronuclei. Differences in the DNA content between pronuclei of the parthenogenones (when there are two in one parthenogenone) were observed beginning with the 10th h after hyaluronidase treatment.The DNA content in the nuclei of the second polar bodies (PB) of embryos increased slowly between the 8th and 22nd hour after insemination, up to an overall value of 1.4 C. That of the nuclei of the polar bodies of parthenogenones accompanied the synthesis of DNA in pronuclei to the 10th hour after hyaluronidase treatment, up to an overall value of 1.4 C.     

Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.     

Changes of Cytochemical Markers in the Conjunctival and Corneal Epithelium After Corneal Debridement

1. The aim of this study was to determine the epithelial changes of the conjunctiva and cornea up to 7 days after corneal debridement and the changes highlighted included (1) proliferation, (2) production of growth factor, (3) changes in calcium binding protein marker, (4) production of cytokine, and (5) maturity of the regeneration corneal epithelium.2. The cytochemical changes of the corneal and conjunctival epithelia of rabbit were analyzed up to 7 days after debridement.3. An increase in proliferating cell nuclear antigen (PCNA) was observed in the limbal epithelia 12 hr after lesion and reached a peak by 48 hr.4. Some proliferating limbal cells also contained epidermal growth factor (EGF) beginning 24 hr after injury. The early limbal cell proliferation and the EGF production and their persistence until 7 days after lesion were likely involved with the process of regeneration.5. Other positive markers appeared after lesion included tumor necrosis factor (TNF) and calcium binding proteins S100A and S100B, which appeared mainly within the first 48 hr after lesion and then started to decline. The short appearance and the relatively small quantity of TNF indicated that this cytokine was probably not very important in the repair process and its appearance might be related to the injury induced. The presence of S100A and S100B could be associated with both cell death after injury and the proliferation of new epithelium.6. The cornea epithelium was still immature 7 days after lesion in that it still contained cytokeratin.7. In conclusion, the critical hours of peak conjunctival and corneal changes after corneal debridement were in the first 2 days.     

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation.

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.     

Ploidy levels and DNA synthesis in fat body cells of the adult mosquito,Aedes aegypti: The role of juvenile hormone

Adult females of the mosquito Aedes aegypti showed two cycles of DNA replication in the fat body based on microspectrophotometric measurement of changes in nuclear DNA. The first cycle began after emergence and resulted in 80% of diploid fat body cells becoming tetraploid and 20% becoming octoploid by the end of the third day. The second replication cycle occurred 48–72 h after a blood meal and resulted in an increase in octoploid nuclei to 67% Topical application of juvenile hormone or methoprene to abdomens isolated at emergence stimulated an increase in ploidy levels above that normally seen in situ. Synthesis of DNA, estimated by incorporation of injected [³H]-thymidine, rose after emergence and remained high for 2 days. Synthesis increased again after a blood meal, reached a peak by 6 h, and returned to low levels by 24 h after the meal. The timing of DNA synthesis and a measurable increase in ploidy were temporally separated. The ploidy increase, but not DNA synthesis, was correlated with increases in juvenile hormone levels.     

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B-A transition in solution

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nanodevice applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40°C by SWNTs but no change for AT-DNA. SWNTs can induce B–A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B–A transition in solution and they bind to the DNA major groove with GC preference.     

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine.

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

Assessment of hemapoietic system reaction of mice when exposed to pulsed magnetic field

The focus of the study was on the reaction of by the haemopoietic system of mice subjected to impulse magnetic field. The source of the impulse magnetic field was the Shakhparonov's generator. The animals used in the experiments were mice of two strains--CBA, C57B1/6 and white non-inbred mice. These animals were exposed to impulse magnetic field during 1, 3 and 7 days. Animals were examined twice: immediately after the termination of exposure and 24 h later. The following effects were observed in the course of the experiments: an increase in the number of bone marrow cells right after the exposure termination; an increase in the number of proliferation pool cells with the increase in their mitotic activity; 1 day after the exposure termination the number of bone marrow cells was restored to the initial values, or even it decreased; the above listed bone marrow changes led to the increase in the number of peripheral blood leucocytes in 1 day after the termination of exposure. The increase of leukocyte counts was not accompanied with changes in peripheral blood cell composition. It was suggested that exposure to impulse magnetic field increases the rates of cell cycle, the cell differentiation and the maturation.     

Fluorometric determination of radiation damages to leukocyte DNA by Birnboim and Jevcak's method

Fluorometric measurements of the levels of radiation injury to DNA of leucocyte lysates were made on the basis of the estimates of alkaline denaturation rate obtained by the method of Birnboim and Jevcak. Leucocytes of human blood in vitro and rats were exposed to doses of 0.5, 1, 1.5, 3, 6 and 9 Gy. It was shown that the percentage of double-stranded DNA of leucocytes of irradiated human blood decreased with the increase in the radiation dose while that of exposed rats decreased immediately after irradiation and was normalized after 20 h.     

Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice

Is the epithelial lining of the mammalian gastrointestinal (GI) tract a tight barrier against the uptake of ingested foreign DNA or can such foreign DNA penetrate into the organism? We approached this question by pipette-feeding circular or linearized double-stranded phage M13 DNA to mice or by adding M13 DNA to the food of mice whose fecal excretions had previously been shown to be devoid of this DNA. At various post-prandial times, the feces of the animals was tested for M 13 DNA sequences by Southern or dot blot hybridization or by the polymerase chain reaction (PCR). On Southern blot hybridization, the majority of M13 DNA fragments were found in the size range between < 200 and 400 by (base pairs). For the PCR analysis, synthetic oligodeoxyribonucleotide primers were spaced on the M13 DNA molecule such that the sizes of the persisting M13 DNA fragments could be determined. We also extracted DNA from whole blood or from sedimented blood cells of the animals at different times after feeding M t3 DNA and examined these DNA preparations for the presence of M13 DNA by dot blot hybridization or by PCR. M13 DNA fragments were found between 1 and 7 h postprandially in the feces of mice. By PCR analysis, fragments of 712, 976, and 1692 by in length were detected. In DNA from blood, M13 DNA fragments of up to 472 by were found by PCR between 2 and 6 h after feeding. Dot blot or Southern blot hybridization revealed M13 DNA at 2 and 4 h, but not at 1, 8 or 24 h after feeding. This DNA was shown to be DNase sensitive. M13 DNA was found both in blood cells and in the serum. A segment of about 400 by of the DNA amplified by PCR from feces or blood was analyzed for its nucleotide sequence which was found to be identical to that of authentic M13 DNA, except for a few deviations. M13 DNA could not be detected in the feces or in the blood of the animals prior to feeding or prior to 1 h and later than 7 h after feeding. These controls attest to the validity of the results and also argue against the possibility that the murine GI tract had been colonized by phage M13. Moreover, M13 DNA-positive bacterial colonies were never isolated from the feces of animals that had ingested M13 DNA. The results of reconstitution experiments suggested that 2 to 4% of the orally administered M13 DNA could be detected in the GI tract of mice. A proportion of about 0.01% to 0.1% of the M13 DNA fed could be retrieved from the blood.