Detailed cell cycle analysis in human lymphocytes; Application to γ-irradiated cells

Summary A method based on BrdU incorporation for analyzing in detail the kinetics of the cell cycle is described. The S phase has been subdivided into five subphases, each recognizable by their BrdU incorporation pattern at metaphase. The method can be useful for the study of abnormal cell cycles, and may have particular application in mutagenesis studies concerning the various subphases of the S phase, without using synchronization techniques. An application of the method is described, showing that -irradiation, during the course of the S phase, leads to a lack of cells which were in early S phase at the time of irradiation. This finding can be related either to a higher lethality at this stage of the cell cycle or to a delay in completion of DNA replication after irradiation.Hoider of a C.E.C. scholarship     

Immunomodulatory and cytoprotective role of RP-1 in γ-irradiated mice

RP-1 has been reported to provide protection against lethal -irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 ± 0.224 and 4.9 ± 0.057 mM Fe²⁺) as compared to control (6.29 ± 0.733 mM Fe²⁺) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G₂ delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G₂ delay and elicited significant (p < 0.05) recovery in S phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.     

Drug—induced alterations in the ultrastructural organization of Histoplasma capsulatum and Blastomyces dermatitidis

Aspects of the fine structure as seen in thin section of yeastlike cells ofHistoplasma capsulatum andBlastomyces dermatitidis exposed to polyenic antibiotics are described and illustrated by electron micrographs. The exposure of log phase yeastlike cells to minimal fungicidal concentrations of both amphotericin B (Fungizone) and hamycin resulted in detectable alterations of the plasma membrane, and, to a lesser extent, the mitochondria. WithH. capsulatum, ultrastructural changes were observed to occur within 1 h exposure to amphotericin B. Marked degenerative changes and plasmolysis were observed to occur within 6 hrs exposure of the yeastlike cells to both polyenes. The observed changes in ultrastructural appearance are compatible with the concept of binding of the polyene with membrane sterol and subsequent damage due to alterations of permeability.     

Caspase-3 antisense oligodeoxynucleotides inhibit apoptosis in γ-irradiated human leukemia HL-60 cells

To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODNs) on apoptosis, we designed four ASODNs targeting different regions of caspase-3 mRNA and transfected them into human leukemia HL-60 cells. The transfected cells were given 10 Gy γ-irradiation followed by incubation for 18 h and measurement of apoptosis and caspase-3 expression. Our results showed that ASODN-2 targeting the 5′ non-coding region of sites –62 to –46, and ASODN-3 targeting the 5′ coding region of sites –1 to 16, both reduced apoptosis measured by gel electrophoresis and flow cytometry. Hoechst 33258 staining and TUNEL assay revealed that apoptotic indexes in the ASODN-2 and ASODN-3 groups were significantly lower than those in the untransfected and mismatched oligodeoxynucleotide (MODN) groups. Immunocytochemistry, Western blotting and RT-PCR showed that expression levels of caspase-3 protein and mRNA in both ASODN-2 and ASODN-3 groups were decreased compared with those in the untransfected and MODN groups. In conclusion, caspase-3 mRNA ASODNs can inhibit γ-radiation-induced apoptosis of HL-60 cells and reduce expression of caspase-3 protein and mRNA. The results suggest that antisense approach may be useful for therapeutic treatment of certain neurodegenerative diseases in which apoptosis is involved. The work was supported by a grant from the National Natural Science Foundation of China (No. 39880008).     

Molecular activation of NF-κB, pro-inflammatory mediators, and signal pathways in γ-irradiated mice

The effects of γ-irradiation on inflammatory gene expression, including NF-κB activation, in the kidney of C57/BL6 mice exposed to 1–9 Gy doses of ⁶⁰Co γ-irradiation. Radiation enhanced the NF-κB activation and oxidative stress caused a dose-dependent disruption in the redox balance. The significance of this study is the new molecular information gained on γ-irradiation effects through the activation of pro-inflammatory genes by NF-κB via the MAPK signaling pathway. Considering the exquisite sensitivity of NF-κB and other pro-inflammatory mediators to the redox status, we conclude that the activation of inflammatory processes by irradiation is likely initiated by increased oxidative stress.     

Immunofluorescence and ultrastructural localization of β2-microglobulin in human renal allografts

Summary The localization of 2-microglobulin (2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, 2m was not observed in the normal kidneys, 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of 2m and its metabolism in the tubular epithelium.     

Structure-function relationships in human α- and γ-thrombins

Summary Human pro-coagulant -thrombin may be proteolyzed under controlled conditions to the non-coagulant - and -thrombin forms. These derivative forms nonetheless retain esterase and amidase activities with small substrates as well as several other thrombin functions. Structurally, human -thrombin consists of three non-covalently associated fragments which retain structural integrity as measured by several spectroscopic criteria as well as enzymatic function. The protein folding characteristics of three-chain -thrombin indicate that each fragment (domain) contains sufficient information to result in a correct renaturation of protein conformation. Those subtle structural differences which distinguish - from -thrombin are most likely the obstructions to fibrinogen binding which account for the loss of clotting activity.     

Antiatherogenic and radioprotective role of folic acid in whole body γ-irradiated mice

Free radical mediated oxidative damage is one of the prime factors for atherogenic changes in humans. We have shown that the folic acid administration reduced the risk of the atherogenic factors induced by γ -radiation. Folic acid administration prevented the radiation induced increase in the plasma lipoprotein lipase activity and also prevented the radiation-induced increase in the hepatic cholesterol and triglycerides levels. These results indicate the role of folic acid as an antiatherogenic agent. Further, we also report the radioprotective property of folic acid as demonstrated by reduction in the radiation induced membrane damage as measured by lipid peroxidation products and DNA damage, which was measured by alkaline comet assay.     

Effect of aphidicolin on de novo DNA synthesis,DNA repair and cytotoxicity in γ-irradiated human fibroblasts. Implications for the enhanced radiosensitivity in ataxia telangiectasia

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase α and consequently of de novo DNA synthesis in human cells. We report here that in γ-irradiated normal human cells, aphidicolin (at 5 μg/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. γ-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase α is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.     

Relationship between DNA chain growth termination and replicon sizes in γ-irradiated mouse cells

Unirradiated mouse leukemic L5178Y cells were pulse-labeled with [³H]thymidine continuously for up to 150 minutes. The resulting DNA fiber autoradiograms showed a continuously increasing fiber length up to 70 μm. However, in cells irradiated with 500 to 2000 rad of γ-rays, the resulting DNA fiber lengths leveled off at approximately 35 μm. Independent experiments to estimate replicon size by measuring the center-to-center distance in tandem tracks yielded a value of 48 μm. These results are consistent with a model proposing that the average replicon size is equal to √2 times the plateau value of the average length of labeled DNA measured in irradiated cells.     

The γ3 chain of laminin is widely but differentially expressed in murine basement membranes: expression and functional studies

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.     

Biochemical changes involved in stress response and ripening behaviour of γ-irradiated mango fruit

The stress response of ripening mango fruits treated in the pre-climacteric phase with radiation doses of 0.75, 1.25 and 1.75 kGy was investigated. L-Phenylalanine ammonialyase, polyphenol oxidase, peroxidase and catalase activities were determined and significant differences were observed. Differences in the patterns of total phenolics, flavanols and proteins were also observed. Malic enzyme activity was used as an indicator of the ripening stage (climacteric rise, climacteric peak and post-climacteric phase) as well as a measure of the effect of γ-irradiation on fruit ripening. It seems that radiation treatment causes a stress condition in the fruit which, depending on the dose, may lead to browning of the tissue or necrotic decay.     

An ultrastructural study of mitosis and cytokinesis in normal ‘resting’ human breast

Summary The parenchyma of the normal resting human breast was examined by electron microscopy to characterize the cells undergoing mitosis and the mechanism by which the normal tissue architecture is maintained during this process. In this study of 112 mitotic cells, it was found that the mitotic cells were luminally positioned, polarised epithelial cells with no evidence of myoepithelial cell division. Ultrastructurally, the nuclear and cytoplasmic changes were consistent with previous reports of mitosis in other tissues. However, unlike all previous reports, two specific orientations of the nuclear spindle and thus the planes of cytokinesis were observed. In a few cases the spindle formed parallel to the lumen and division resulted in two luminally positioned daughter cells. However, in the majority of mitotic cells the spindle was approximately at right angles to the lumen and this orientation resulted in a luminally and a basally positioned daughter cell. It is proposed that the abnormally positioned basal daughter cell could develop into a myoepithelial cell or undergo deletion (apoptosis). Thus the two orientations of mitosis may explain the mechanism by which the epithelial and myoepithelial cell populations were maintained by a single progenitor cell without disrupting the integrity of the tissue architecture.     

Reduced tonsillar expression of human β-defensin 1, 2 and 3 in allergic rhinitis

Airway infections are known to cause exacerbations of allergy and asthma. Tonsils constitute a primary site for microbial recognition and triggering of the immune system in the airways. Human β-defensins (HBDs) are antimicrobial peptides with an important role in this defense. Our aim was to investigate HBD1-3 in tonsillar tissue and their potential role in allergic rhinitis (AR). Tonsils, obtained from patients with AR and non-allergic controls, and isolated tonsillar CD4(+), CD8(+) and CD19(+) lymphocytes were analyzed for HBD1-3 expression using real-time RT-PCR and/or immunohistochemistry. Tonsillar tissue, mixed tonsillar lymphocytes and airway epithelial cells (AECs) were cultured with or without IL-4, IL-5, IL-13 or histamine followed by measurements of HBD1-3 release using ELISA. HBD1-3 were present in tonsillar tissue, including epithelial, CD4(+), CD8(+) and CD19(+) cells. The expression was reduced in allergic compared to healthy tonsils. Stimulation of AECs with IL-4, IL-5 and histamine down-regulated the HBD release, whereas no effects were seen in cultured tonsils or lymphocytes. This study demonstrates presence of HBD1-3 in tonsils and that the levels are reduced in patients with AR. Together with the down-regulation of HBDs in epithelial cells in the presence of allergic mediators suggest that AR patients have an impaired antimicrobial defense that might make them more susceptible to respiratory tract infections.     

Identification, localization and characterization of the human γ-synuclein gene

We have identified and characterized a new member of the human synuclein gene family, γ-synuclein (SNCG). This gene is composed of five exons, which encode a 127 amino acid protein that is highly homologous to α-synuclein, which is mutated in some Parkinson’s disease families, and to β-synuclein. The γ-synuclein gene is localized to chromosome 10q23 and is principally expressed in the brain, particularly in the substantia nigra. We have determined its genomic sequence, and established conditions for sequence analysis of each of the exons. The γ-synuclein gene, also known as BCSG1, was recently found to be overexpressed in advanced infiltrating carcinoma of the breast. Our survey of the EST database indicated that it might also be overexpressed in an ovarian tumor. Received: 6 February 1998 / Accepted: 8 April 1998