Formation of a DNA monofunctional cis-platinum adduct cross-linking the intercalating drug N-methyl-2,7-diazapyrenium.

Our purpose was to better understand the mutual influence of cis-diamminedichloroplatinum (II) (cis-DDP) and intercalating drugs in their interactions with DNA. The present study deals with the intercalating drug N-methyl-2,7-diazapyrenium (MDAP). Two sets of experiments have been performed. In one set, the reaction between cis-DDP and nucleic acid was carried out in the presence of MDAP. The main adduct is a guanine residue chelated by platinum to a MDAP residue. It has the same spectroscopic properties as the synthesized compound cis-[Pt (NH3)2 (N7-d-guanosine) (N7-MDAP)] , the structure of which has been determined by 1H NMR. This adduct was only formed with double-stranded nucleic acids which reveals the importance of DNA matrix in orienting favorably the reactants. In the second set of experiments, the triamine complex cis-[Pt(NH3)2 (MDAP)CI]++ was reacted with the nucleic acids. At molar ratios drug over nucleotide residue equal or less than 0.10, all the added triamine complexes bind by covalent coordination to double-stranded nucleic acids. With natural DNA, the major adduct is cis-[Pt(NH3)2(d-guanosine) (MDAP)] . Thus the same adduct is formed on one hand in the reaction between DNA, MDAP and cis-DDP and on the other hand in the reaction between the triamine complex and DNA. The triamine complex offers the possibility to study the biological role of the new adduct.     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

Cutting edge: Deficiency of macrophage migration inhibitory factor impairs murine airway allergic responses

Increased levels of macrophage migration inhibitory factor (MIF) in serum, sputum, and bronchioalveolar lavage fluid (BALF) from asthmatic patients and time/dose-dependent expression of MIF in eosinophils in response to phorbol myristate acetate suggest the participation of MIF in airway inflammation. In this study, we examined inflammation in OVA-sensitized mouse lungs in wild-type and MIF-deficient mice (MIF(-/-)). We report increased MIF in the lung and BALF of sensitized wild-type mice. MIF(-/-) mice demonstrated significant reductions in serum IgE and alveolar inflammatory cell recruitment. Reduced Th1/Th2 cytokines and chemokines also were detected in serum or BALF from MIF(-/-) mice. Importantly, alveolar macrophages and mast cells, but not dendritic cells or splenocytes, from MIF(-/-) mice demonstrated impaired CD4+ T cell activation, and the reconstitution of wild-type mast cells in MIF(-/-) mice restored the phenotype of OVA-induced airway inflammation, revealing a novel and essential role of mast cell-derived MIF in experimentally induced airway allergic diseases.     

Comparison Between poly(dG-dC)·poly(dG-dC) and DNA Modified by cis-diamminedichloroplatinum (II): Immunological and Spectroscopic Studies

Abstract

The importance of the base composition and of the conformation of nucleic acids in the reaction with the drug cis-diamminedichloroplatinum(II) has been studied by competition experiments between the drug and several double-stranded polydeoxyribonucleotides. Binding to poly(dG)·poly(dC) is larger than to poly (dG-dC)·poly(dG-dC). There is no preferential binding in the competition between poly(dG-dC) ·poly(dG-dC), poly(dA-dC) ·poly(dG-dT) and poly(dA-dG)·poly(dC-dT). In the competition between poly(dG-dC) ·poly (dG-dC) (B conformation) and poly(dG-br⁵dC) ·poly(dG-br⁵dC) (Z conformation), the drug binds equally well to both polynucleotides. In natural DNA, modification of guanine residues in (GC)_n·(GC)_nsequences by the drug has been revealed by the inhibition of cleavage of these sequences by the restriction enzyme BssHII. By means of antibodies to platinated poly(dG-dC), it is shown that some of the adducts formed in platinated poly(dG-dC) are also formed in platinated pBR322 DNA. The type of adducts recognized by the antibodies is not known. Thin layer chromatography of the products after chemical and enzymatic hydrolysis of platinated poly(dG-dC) suggests that interstrand cross-links are formed. Finally, the conformations of poly(dG-m⁵dC) modified either by cis-diamminedichloroplatinum(II) or by trans-diammine- dichloroplatinum(II) have been compared by circular dichroism. Both the cis-isomer and the trans-isomer stabilize the Z conformation when they bind to poly(dG-m⁵dC) in the Z conformation. When they bind to poly(dG-m⁵dC) in the B conformation, the conformations of poly(dG-m⁵dC) modified by the cis or the trans-isomer are different. Moreover, the cis-isomer facilitates the B form-Z form transition of the unplatinated regions while the trans-isomer makes it more difficult.     

Magnetic Resonance Angiography Signal Intensity as a Marker of Hemodynamic Impairment in Intracranial Arterial Stenosis

Background

Intracranial arterial stenosis (ICAS) is the predominant cause of ischemic stroke and transient ischemic attack in Asia. Change of signal intensities (SI) across an ICAS on magnetic resonance angiography (MRA) may reflect its hemodynamic severity.

Methods

In-patients with a symptomatic single ICAS detected on 3D time-of-flight MRA were recruited from 2 hospitals. Baseline and 1-year follow-up data were collected. Signal intensity ratio (SIR) [ =  (mean post-stenotic SI -mean background SI)/(mean pre-stenotic SI - mean background SI)] was evaluated on baseline MRA to represent change of SIs across an ICAS. Acute infarct volume was measured on baseline diffusion-weighted images (DWI). Relationships between SIR and baseline characteristics as well as 1y outcomes were evaluated.

Results

Thirty-six subjects (86.1% males, mean age 55.0) were recruited. Overall, mean SIR was 0.84±0.23. Mean SIRs were not significantly different between the 23 (63.9%) anatomically severe stenoses and the 13 (36.1%) anatomically moderate stenoses (0.80±0.23 versus 0.92±0.21, p = 0.126). SIR was significantly, linearly and negatively correlated to acute infarct volume on DWI (Spearman correlation coefficient −0.471, p = 0.011). Two patients (5.6%) had recurrent ischemic strokes at 1y, not related to SIR values.

Conclusions

Change of signal intensities across an ICAS on MRA may reflect its hemodynamic and functional severity. Future studies are warranted to further verify the relationships between this index and prognosis of patients with symptomatic ICAS.     

Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.     

Effects of a moderate-intensity static magnetic field and adriamycin on K562 cells

The aim of this study was to investigate whether a moderate‐intensity static magnetic field (SMF) can enhance the killing effect of adriamycin (ADM) on K562 cells, and to explore the effects of SMF combined with ADM on K562 cells. We analyzed the metabolic activity of cells, cell cycle distribution, DNA damage, change in cell ultrastructure, and P‐glycoprotein (P‐gp) expression after K562 cells were exposed continuously to a uniform 8.8 mT SMF for 12 h, with or without ADM. Our results showed that the SMF combined with ADM (25 ng/ml) significantly inhibited the metabolic activity of K562 cells (P < 0.05), while neither ADM nor the SMF alone affected the metabolic activity of these cells. Cell ultrastructure was altered in the SMF + ADM group. For example, cell membrane was depressed, some protuberances were observable, and vacuoles in the cytoplasm became larger. Cells were arrested at the G2/M phase and DNA damage increased after cells were treated with the SMF plus ADM. ADM also induced the P‐gp expression. In contrast, in the SMF group and SMF + ADM group, the P‐gp expression was decreased compared with the ADM group. Taken together, our results showed that the 8.8 mT SMF enhanced the cytotoxity potency of ADM on K562 cells, and the decrease in P‐gp expression may be one reason underlying this effect. Bioelectromagnetics 32:191–199, 2011. © 2010 Wiley‐Liss, Inc.     

The gene conferring susceptibility to spot blotch caused by <Emphasis Type="Italic">Cochliobolus sativus</Emphasis> is located at the <Emphasis Type="Italic">Mla</Emphasis> locus in barley cultivar Bowman

Key message

We identified, fine mapped, and physically anchored a dominant spot blotch susceptibility gene Scs6 to a 125 kb genomic region containing the Mla locus on barley chromosome 1H.

Abstract

Spot blotch caused by Cochliobolus sativus is an important disease of barley, but the molecular mechanisms underlying resistance and susceptibility to the disease are not well understood. In this study, we identified and mapped a gene conferring susceptibility to spot blotch caused by the pathotype 2 isolate (ND90Pr) of C. sativus in barley cultivar Bowman. Genetic analysis of F₁ and F₂ progeny as well as F₃ families from a cross between Bowman and ND 5883 indicated that a single dominant gene (designated as Scs6) conferred spot blotch susceptibility in Bowman. Using a doubled haploid (DH) population derived from a cross between Calicuchima-sib (resistant) and Bowman-BC (susceptible), we confirmed that Scs6, contributed by Bowman-BC, was localized at the same locus as the previously identified spot blotch resistance allele Rcs6, which was contributed by Calicuchima-sib and mapped on the short arm of chromosome 1H. Using a genome-wide putative linear gene index of barley (Genome Zipper), 13 cleaved amplified polymorphism markers were developed from 11 flcDNA and two EST sequences and mapped to the Scs6/Rcs6 region on a linkage map constructed with the DH population. Further fine mapping with markers developed from barley genome sequences and F₂ recombinants derived from Bowman?×?ND 5883 and Bowman?×?ND B112 crosses delimited Scs6 in a 125 kb genomic interval harboring the Mla locus on the reference genome of barley cv. Morex. This study provides a foundational step for further cloning of Scs6 using a map-based approach.

     

The incorporation of acetate,stearate and d(?)-β-hydroxybutyrate into milk fat by the isolated perfused mammary gland of the goat

1. Mammary glands of lactating goats were perfused for 12.5-15hr. with heparinized whole blood and infused with a substrate mixture of glucose, acetate and amino acids (and sometimes chylomicra) containing either [1-(14)C]acetate, d(-)-beta-hydroxy[1-(14)C]butyrate or [U-(14)C]stearate. 2. There was a substantial net uptake of acetate by the glands and transfer of radioactivity into milk fat. Acetate was extensively utilized for the synthesis of milk fatty acids of chain length up to C(14) and to a smaller extent for the synthesis of palmitate. 3. There was a small and variable net uptake of stearate and beta-hydroxybutyrate and negligible oxidation of these substrates. However, tissue uptake was demonstrated by a substantial fall in specific radioactivity across the glands and an extensive transfer of radioactivity into milk fatty acids. 4. With beta-hydroxybutyrate the labelling of milk fat was very similar to that with acetate, but the distribution of radioactivity suggested a cleavage into C(2) fragments of about 40%. 5. Labelled stearate gave rise to highly labelled stearate and oleate in the milk fat. Small amounts of radioactivity were detected in stearate of plasma triglycerides and oleate of plasma free fatty acids. 6. In experiments where there was a decline in milk-fat secretion late in perfusion, the milk fatty acids showed a marked decline in the proportion of stearate and oleate and a rise in the proportion of myristate and palmitate. This did not occur in experiments where milk-fat secretion was maintained at a higher level. 7. The present results confirm that there is a large pool of long-chain fatty acids in mammary tissue that can act as an endogenous source of these substrates.     

Up‐regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR‐296‐5p

LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR‐296‐5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT‐PCR. The targeting relationship between miR‐296‐5p and ZFAS1 or USF1 was validated by dual‐luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK‐8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR‐296‐5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up‐regulated in tumour tissues and cells while miR‐296‐5p was significantly down‐regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR‐296‐5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR‐296‐5p.