Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Investigation of the dark interaction between furocoumarins and DNA

The complexes between some furocoumarins and DNA have been studied using various physicochemical techniques. Flow-dichroism measurements data strongly support the intercalation of the planar furocoumarin molecules between two base pairs of duplex DNA. The equilibrium dialysis and spectrophotometric data show relatively low values of the association constants of the complexes and a small number of molecules able to intercalate in DNA, thus indicating that furocoumarins have a relatively low affinity for DNA in the complex formation. The biological and photobiological consequences connected with these results are discussed.The binding curves obtained using some polynucleotides and various DNA samples having different composition with regard to base pairs, have shown that the regions of the macromolecule having alternate sequences of purine and pyrimidine represent sites useful for intercalation. No preference has been observed for A-T or G-C.     

Chromosomal damage induced by furocoumarins and UVA in hamster and human cells including cells from patients with ataxia telangiectasia and xeroderma pigmentosum

The comparative photosensitizing effects to near-UV irradiation (UVA) of several naturally occurring furocoumarins, 5-methoxypsoralen (5MOP), psoralen, 8-methoxypsoralen (8MOP) and angelicin in producing chromosome damage in vitro in cells derived from hamster, normal human, ataxia telangiectasia (AT) and xeroderma pigmentosum (XP) patients were studied. In Chinese hamster cells, lethality was greatest with psoralen and least with angelicin; 8MOP and 5MOP were intermediate. 8MOP and 5MOP produced sister-chromatid exchanges with almost equal efficiency and to a larger extent by far than angelicin. In all human cell lines studied 8MOP and 5MOP were similarly effective in the production of sister-chromatid exchanges and chromosomal aberrations. AT and XP cells responded with higher frequencies of sister-chromatid exchanges as well as chromosomal aberrations than normal human cells to 5MOP, 8MOP and angelicin. Evidence is presented which suggests that cell death in Chinese hamster cells following angelicin photosensitization is not clearly related to the production of sister-chromatid exchanges. AT cells were unexpectedly more sensitive to angelicin than normal cells. The presence of 5MOP in some sun-tan preparations is not acceptable in view of the present evidence of its biological activity.     

Rescue by chloramphenicol and rifampicin of UV-irradiated lon, exrA and recA mutants of Escherichia coli K12

The effect of temporary treatment with chloramphenicol or rifampicin on the survival of UV irradiated cells of selected Escherichia coli K12 radiation sensitive mutants was examined. Increased survival resulted for both exrA and recA mutants, and also for the unsuppressed lon mutant, but cells of the parent strain and the recB mutant were not rescued. This contrasts with our earlier finding that after exposure of the bacteria to γ-rays, chloramphenicol treatment rescued the exrA and lon mutants but not the recA mutant. We now report that an exrA recA double mutant was rescued by chlramphenicol after UV radiation, but not after anaerobic ionizing radiation. Inclusion of inhibitors of uvrA governed repair, caffeine and 8-methoxypsoralen (8-MOP), in the incubation medium containing chloramphenicol, did not reduce the rescue of the exrA or recA mutants, although caffeine eliminated rescue of the lon mutant, which was itself unaffected by 8-MOP. However it is concluded that chlormaphenicol rescue of the exrA and recA mutants after UV radiation was not entirely independent of the excision-repair process, since the uvrA recA and uvrA exrA double mutants were not rescued by this treatment.     

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells

AimThe nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr¹⁰]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of ¹²⁵I-[Tyr¹⁴]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr¹⁰]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr¹⁰]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr¹⁰]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors.Main methodsThe NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors.Key findingsIn vlPAG neurons, [Tyr¹⁰]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC₅₀: 8.98 μM) but equi-efficacious as N/OFQ. [Tyr¹⁰]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr¹⁰]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation.Significance[Tyr¹⁰]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.     

Genetic effects of specific DNA lesions in mammalian cells

Accurate dosimetry for chemical mutagens is extremely difficult, and precise manipulation of the frequency of a particular lesion is ordinarily impossible. With 8-MOP plus UVA, however, both are possible because 8-MOP, when photoactivated by one photon of UVA, forms monoadducts whilst crosslinks are formed only if a second photon of light photoactivates the monoadducts. If 8-MOP molecules that are unreacted after a UVA exposure are removed from cells by washing, the effect of a subsequent UVA irradiation can be attributed only to the conversion of monoadducts to DNA interstrand crosslinks. Using this experimental procedure and L5178Y mouse lymphoma cells, we have shown that DNA interstrand crosslinks are at least 10-fold more effective at causing both sister-chromatid exchanges and chromosomal aberrations than are monoadducts. In contrast, crosslinks are no more effective than monoadducts in mutation induction. These experiments identify directly for the first time that a particular chemically induced lesion, DNA interstrand crosslinks, can, like thymine dimers, cause chromosomal aberrations and sister-chromatid exchanges. The results also show that sister-chromatid exchanges can be induced independently of mutations.     

The mortality of MOP3 deficient mice with a systemic functional failure

Summary MOP3 (also known as BMAL1), a master regulator of circadian rhythm, plays important roles in the regulation of cell differentiation and general physical functions. In the present studies, MOP3 deficient mice had significantly reduced body weight and showed remarkable mortality around six months of age. The levels of AST, ALT, BUN, or UREA in the blood of about four month-old MOP3^−/− mice were significantly higher than MOP3^+/− or MOP3^+/+ littermates. However, no apparent pathological changes in the livers, hearts, lungs or kidneys of about four month-old MOP3^−/− mice were observed. In addition, altered levels of white blood cells, lympgocytes, and platelets in peripheral blood of MOP3^−/−mice were detected. The results presented herein with MOP3-deficient mice offered the basic principle for the essential roles of MOP3 in keeping normal survival abilities in mice. This study may have significant clinical impacts on the consideration about the abnormality of circadian rhythms and sleeping disorders caused physical and metabolism dysfunctions as well as the mortality.     

Mechanism of μ-Opioid Receptor-Magnesium Interaction and Positive Allosteric Modulation

In the era of opioid abuse epidemics, there is an increased demand for understanding how opioid receptors can be allosterically modulated to guide the development of more effective and safer opioid therapies. Among the modulators of the μ-opioid (MOP) receptor, which is the pharmacological target for the majority of clinically used opioid drugs, are monovalent and divalent cations. Specifically, the monovalent sodium cation (Na⁺) has been known for decades to affect MOP receptor signaling by reducing agonist binding, whereas the divalent magnesium cation (Mg²⁺) has been shown to have the opposite effect, notwithstanding the presence of sodium chloride. Although ultra-high-resolution opioid receptor crystal structures have revealed a specific Na⁺ binding site and molecular dynamics (MD) simulation studies have supported the idea that this monovalent ion reduces agonist binding by stabilizing the receptor inactive state, the putative binding site of Mg²⁺ on the MOP receptor, as well as the molecular determinants responsible for its positive allosteric modulation of the receptor, are unknown. In this work, we carried out tens of microseconds of all-atom MD simulations to investigate the simultaneous binding of Mg²⁺ and Na⁺ cations to inactive and active crystal structures of the MOP receptor embedded in an explicit lipid-water environment and confirmed adequate sampling of Mg²⁺ ion binding with a grand canonical Monte Carlo MD method. Analyses of these simulations shed light on 1) the preferred binding sites of Mg²⁺ on the MOP receptor, 2) details of the competition between Mg²⁺ and Na⁺ cations for specific sites, 3) estimates of binding affinities, and 4) testable hypotheses of the molecular mechanism underlying the positive allosteric modulation of the MOP receptor by the Mg²⁺ cation.     

Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF₂ receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [³⁵S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [³⁵S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with α_s, α_i2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.     

涉及光疗机制的生物光化学

本文对光生物和光化学的定义,反应机制的类型和光敏化作用等做了阐述。下面例举几个光疗的成果 1.光疗牛皮癣 经常使用的8-甲氧基补骨脂素在UVA的照射下,从基态被激发到三重态。它主要和DNA中的胸腺嘧啶,其次和色氨酸进行光环合加成,形成交联,阻止DNA和RNA的合成,抑制具过度增生 2.血卟啉衍生物(HPD)治癌 HPD有定位于癌组织的能力和光动力作用,可推断病人体内癌部位。 讨论了HPD的光疗机制,和酞菁相比,有各自的优缺点。 3.竹红菌素 主要治疗妇女外阴白色病变和疤痕疙瘩,抑制癌细胞生长。 讨论了竹红菌甲素和乙素及它们的氧化物的结构和活性。 在大于510nm的光照射下,也可抑制癌细胞的生长。列举了竹红菌素的优缺点。     

A novel magnetic biochar efficiently sorbs organic pollutants and phosphate

Biochar derived from agricultural biomass waste is increasingly recognized as a multifunctional material for agricultural and environmental applications. Three novel magnetic biochars (MOP250, MOP400, MOP700) were prepared by chemical co-precipitation of Fe³⁺/Fe²⁺ on orange peel powder and subsequently pyrolyzing under different temperatures (250, 400 and 700 °C), which resulted in iron oxide magnetite formation and biochar preparation in one-step. The MOP400 was comprised of nano-size magnetite particles and amorphous biochar, and thus exhibited hybrid sorption capability to efficiently remove organic pollutants and phosphate from water. For organic pollutants, MOP400 demonstrated the highest sorption capability, and even much larger than the companion non-magnetic biochar (OP400). For phosphate, magnetic biochars, especially MOP250, demonstrated much higher sorption capability than the companion non-magnetic biochars. No significantly competitive effect between organic pollutant and phosphate was observed. These suggest that the magnetic biochar is a potential sorbent to remove organic contaminants and phosphate simultaneously from wastewater.     

Hypoxia worsens the impact of intracellular triglyceride accumulation promoted by electronegative low-density lipoprotein in cardiomyocytes by impairing perilipin 5 upregulation

Plasma lipoproteins are a source of lipids for the heart, and the proportion of electronegative low density lipoprotein [LDL(−)] is elevated in cardiometabolic diseases. Perilipin 5 (Plin5) is a crucial protein for lipid droplet management in the heart. Our aim was to assess the effect of LDL(−) on intracellular lipid content and Plin5 levels in cardiomyocytes and to determine whether these effects were influenced by hypoxia. HL-1 cardiomyocytes were exposed to native LDL [LDL(+)], LDL(−), and LDL(+) enriched in non-esterified fatty acids (NEFA) by phospholipase A₂ (PLA₂)-mediated lipolysis [PLA₂-LDL(+)] or by NEFA loading [NEFA-LDL(+)] under normoxia or hypoxia. LDL(−), PLA₂-LDL(+) and NEFA-LDL(+) raised the intracellular NEFA and triglyceride (TG) content of normoxic cardiomyocytes. Plin5 was moderately upregulated by LDL(+) but more highly upregulated by LDL(−), PLA₂-LDL(+) and NEFA-LDL(+) in normoxic cardiomyocytes. Hypoxia enhanced the effect of LDL(−), PLA₂-LDL(+) and NEFA-LDL(+) on intracellular TG and NEFA concentrations but, in contrast, counteracted the upregulatory effect of these LDLs on Plin5. Fluorescence microscopy experiments showed that hypoxic cardiomyocytes exposed to LDL(−), PLA₂-LDL(+) and NEFA-LDL(+) have an increased production of reactive oxygen species (ROS). By treating hypoxic cardiomyocytes with WY-14643 (PPARα agonist), Plin5 remained high. In this situation, LDL(−) failed to enhance intracellular NEFA concentration and ROS production. In conclusion, these results show that Plin5 deficiency in hypoxic cardiomyocytes exposed to LDL(−) dramatically increases the levels of unpacked NEFA and ROS.     

Zeolitic imidazolate framework-8 (ZIF-8) doped TiZSM-5 and Mesoporous carbon for antibacterial characterization

Drug resistant bacteria affects millions worldwide and remains a serious threat to health care system. The study reports the first application of hybrid nanocomposites based on zeolitic imidazolate framework-8 (ZIF-8) with MFI structured zeolite Ti-ZSM-5 (TiZ5) and mesoporous carbon (MC). The composite was designated as TiZ5/ZIF-8 and MC/ZIF-8 was studied for antibacterial activity. Bioactive components Zn²⁺ and 2-methyl imidazole present in ZIF-8 was found to exert significant antibacterial effect on Escherchia. coli and Staphyloccocus. No other antibiotic drugs are required. For comparative purpose, Fe-BTC MOF (BTC = 1,3,5‐benzenetricarboxylate) was used as second set of nanoformulations (TiZ5/Fe-BTC and MC/Fe-BTC) but showed a lower antibacterial activity. The phase (X-ray diffraction), texture (BET surface area), coordination (DRS-UV–Vis), and morphology (TEM) was investigated. XRD showed the presence of nanosized ZIF-8 over TiZ5 and MC. Surface area calculation using N₂ adsorption isotherm showed a reduction in the micropore surface area of ZIF-8 from 1148 m²/g to 224 m²/g (80%) and an increased meso surface area from 31 m²/g to 59 m²/g (90%). The mesopore pore volume increased significantly from 0.05 cm³/g to 0.12 m²/g. MC/ZIF-8 showed similar textural modifications. FT-IR spectra and DRS-UV–Vis spectra showed distinct composite formation with TiZ5, while a weak absorption of ZIF-8 observed over MC. TEM revealed the presence of nanocomposite MC/ZIF-8 and TiZ5/ZIF-8 distributed in nanosize ranging between 25 and 50 nm. TiZ5/ZIF-8 showed the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 0.5 and 1 mg/ml, respectively against E. coli. The MIC and MBC of TiZ5/ZIF-8 against S. aureus were 1 and 2 mg/ml, respectively. MC/ZIF-8 composite had second best antibacterial activity. This study shows that ZIF-8 based composite holds a great potential against E. coli and S. aureus.     

Mechanism of the gas phase reactions of the C5H5Ni and C5H5Co ions with substituted pyridines. A combined experimental and theoretical study

Different products have been observed in the reactions of C₅H₅Co⁺ and C₅H₅Ni⁺ ions with halogen-substituted pyridines (XPy) that have been studied by ion trap mass spectrometry (ITMS) techniques. In particular, an addition product C₅H₅M(XPy)⁺ and a product ion C₅H₄M(Py)⁺ corresponding to a loss of a HX molecule (X = F, Cl, Br) have been detected. The relative yield of these products is determined by the nature of the metal and by the nature and position of the halogen on the pyridine ring. A computational study at the DFT level on model-systems formed by 2-fluoro and 2-bromopyridine reacting either with the C₅H₅Ni⁺ or the C₅H₅Co⁺ ion has been carried out. This study shows the existence of a general mechanistic pattern. The rate-determining step of this mechanism is the migration of the halogen from the pyridine ring to the metal. A final hydrogen abstraction step carried out by the halogen leads to the expulsion of a HX molecule. The existence of avoided crossings between surfaces of different multiplicities (ground and first excited state) allows the system to follow lower energy reaction pathways. The barrier determined for the reactions involving 2-bromopyridine is significantly lower than that found for 2-fluoropyridine. This is mainly due to the poor migrating/leaving character and low polarizability of fluorine compared to that of bromine.     

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

T4 RNA ligase 2 (Rnl2) repairs 3′-OH/5′-PO₄ nicks in duplex nucleic acids in which the broken 3′-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2–(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the 5′-PO₄ of the nick to form an activated AppN– intermediate (step 2); and attack by the nick 3′-OH on the AppN– strand to form a 3′–5′ phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2–AMP. For substrates with correctly base-paired 3′-OH nick termini, k_step2 was fast (9.5 to 17.9 sec⁻¹) and similar in magnitude to k_step3 (7.9 to 32 sec⁻¹). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3′-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3′-OH elicited severe decrements in the rate of 5′-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3′-terminal ribonucleoside at the nick for optimal 5′-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.     

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb ⁵. Lpb ⁵ pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb ⁵ pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.     

Conformation of the peptide antibiotic — histatin 8 in aqueous and non aqueous media

Histatin 8 (Lys¹-Phe-His-Glu-Lys⁵-His-His-Ser-His-Arg¹⁰-Gly-Tyr¹²)belongs to a group of related neutral and basic histidine richpeptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of thispeptide has been examined by ¹H and ¹³C 2D-NMR in DMSO-d₆, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined by MARDIGRAS employing the RANDMARDI approach. In both DMSO-d₆ and water, the peptide is seen to adopt a -pleated sheet, while HFA induces an -helix structure. The role of these structures in its mechanism of action has been explained.     

Photopheresis with UV-A light and 8-methoxypsoralen leads to cell death and to release of blebs with anti-inflammatory phenotype in activated and non-activated lymphocytes

Background: Extracorporeal photopheresis is a therapy for treatment of autoimmune diseases, cutaneous T-cell lymphoma, organ graft rejection as well as graft-versus-host diseases. The exact mechanism how the combination of 8-methoxypsoralen plus UV-A irradiation (PUVA) acts is still unclear. We investigated the cell death of activated and non-activated lymphocytes after PUVA treatment as well as the rate of released blebs and their antigen composition. Results: In presence of 8-MOP, UV-A light highly significantly increased the cell death of activated lymphocytes. The same was observed to a lesser extent in non-activated cells. Blebs derived from activated lymphocytes after PUVA treatment showed the highest surface exposition of phosphatidylserine. These blebs also displayed a high exposure of the antigens CD5 and CD8 as well as a low exposure of CD28 and CD86. Conclusion: PUVA treatment exerts anti-inflammatory effects by inducing apoptosis and apoptotic cell-derived blebs with immune suppressive surface composition.     

8‐Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide‐induced inflammatory injury via suppressing JAK/STAT and NF‐κB pathway

Bovine mastitis is the most common disease in dairy cattle. Bacterial infections are the main cause of mastitis. Lipopolysaccharide (LPS), a major structural component of the cell wall of Escherichia coli, is a good inducer used to replicate inflammation models. 8‐Methoxypsoralen (8‐MOP), a formerly considered photosensitizing agent, has been used in immunotherapy. This study investigated the protective effects of 8‐MOP on LPS‐induced inflammatory injury in bovine mammary epithelial cells (BMECs). LPS treatment (50 μg/mL for 12 hr) caused a decrease in cell viability, morphological damage, and cell apoptosis. Pretreatment with 8‐MOP at concentrations of 25 and 50 μg/ml significantly attenuated LPS‐induced inflammation in BMECs. qRT‐PCR analysis revealed that the messenger RNA expression of inflammatory cytokines and chemokine (interleukin‐1β [IL‐1β], IL‐6, tumor necrosis factor‐α, and IL‐8) was suppressed by 8‐MOP in LPS‐stimulated BMECs. Western blot analysis showed that 8‐MOP could also reduce the protein levels of cyclooxygenase‐2 and promote the translocation of high‐mobility group box 1 from the nucleus to cytoplasm. Furthermore, the anti‐inflammatory property of 8‐MOP was mediated by inhibiting nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation and STAT1 phosphorylation. Taken together, 8‐MOP could protect cells from inflammatory injury induced by LPS, and may be a potential agent against bovine mastitis.     

Wheat Neocentromeres Found in F1 Triticale × Tritordeum Hybrids (AABBRH<Superscript>ch</Superscript>) After 5-Azacytidine Treatment

The DNA hypomethylation effect of 5-azacytine (5-AC; a cytosine analog) is widely known. This agent has been used for rRNA gene expression studies of Triticeae amphiploids and hybrids regarding rye rRNA genes suppression caused by the wheat nucleolar dominance phenomenon. However, this situation is reverted by 5-AC treatment which activates rye rRNA gene expression as it has been intensively observed in triticale. For nucleolar dominance studies, we produced F1 multigeneric hybrids (AABBRH^ch; 2n = 6x = 42) from crosses between the triticale cultivar ‘Corgo’ (AABBRR; 2n = 6x = 42) and the tritordeum cultivars HT9 and HT31 (AABBH^chH^ch; 2n = 6x = 42). The hybrid seeds were germinated in a low concentration of 5-AC (treatment) and in distilled water (nontreated control plants). Silver nitrate staining performed in one 5-AC-treated F1 hybrid revealed a reduced number of interphase cells with seven nucleoli, metaphases with eight Ag-NORs, and neocentromeres in the long arm of three wheat chromosomes. Nontreated hybrids presented six Ag-NORs per mitotic metaphase cell and a maximum of six nucleoli per interphase because of the 1R Ag-NOR suppression. No neocentromere was found in the control F1 hybrid plants. Both treated and nontreated seedlings were subsequently evaluated by fluorescent in situ hybridization performed with genomic and repetitive DNA probes to identify H^ch and rye genomes, to confirm Ag-NORs location, and to detect inactive rDNA loci. DAPI counterstaining was also helpful for the detection of neocentromeres in the long arm of three wheat chromosomes. This study allowed us to suggest that 5-AC treatment specifically induced wheat neocentromeres in the F1 multigeneric triticale × tritordeum hybrids.