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Nanoscale soft-X-ray microscopy is a powerful analysis tool in biological, chemical, and physical sciences. To enhance its probe sensitivity and leverage multimodal soft-X-ray microscopy, precise achromatic focusing devices, which are challenging to fabricate, are essential. Here, we develop an ultracompact Kirkpatrick-Baez (ucKB) mirror, which is ideally capable of high-performance nanofocusing for broadband-energy X-rays. We apply our advanced fabrication techniques and short-focal-length strategy to realize diffraction-limited focusing over the entire soft-X-ray range. We achieve a focus size of 20.4 nm at 2 keV, representing a significant improvement in achromatic soft-X-ray focusing. The ucKB mirror extends soft-X-ray fluorescence microscopy by producing a bicolor nanoprobe with a 1- or 2-keV photon energy. We propose a subcellular chemical mapping method allowing a comprehensive analysis of specimen morphology and the distribution of light elements and metal elements. ucKB mirrors will improve soft-X-ray nanoanalyses by facilitating photon-hungry, multimodal, and polychromatic methods, even with table-top X-ray sources.

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1–induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1–infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome–related tumor development. (Cancer Res 2006; 66(2): 627-31)

Supplementary Method from HIV-1 Vpr Induces DNA Double-Strand Breaks

It is widely recognized that the toxicity of mercury (Hg) is attenuated by the simultaneous administration of selenium (Se) compounds in various organisms. In this study, we revealed the mechanisms underlying the antagonistic effect of sodium selenite (Na2SeO3) on inorganic Hg (Hg2+) toxicity in human hepatoma HepG2 cells. Observations by transmission electron microscopy indicated that HgSe (tiemannite) granules of up to 100 nm in diameter were accumulated in lysosomal-like structures in the cells. The HgSe granules were composed of a number of HgSe nanoparticles, each measuring less than 10 nm in diameter. No accumulation of HgSe nanoparticles in lysosomes was observed in the cells exposed to chemically synthesized HgSe nanoparticles. This suggests that intracellular HgSe nanoparticles were biologically generated from Na2SeO3 and Hg2+ ions transported into the cells and were not derived from HgSe nanoparticles formed in the extracellular fluid. Approximately 85% of biogenic HgSe remained in the cells at 72 h post culturing, indicating that biogenic HgSe was hardly excreted from the cells. Moreover, the cytotoxicity of Hg2+ was ameliorated by the simultaneous exposure to Na2SeO3 even before the formation of insoluble HgSe nanoparticles. Our data confirmed for the first time that HepG2 cells can circumvent the toxicity of Hg2+ through the direct interaction of Hg2+ with a reduced form of Se (selenide) to form HgSe nanoparticles via a Hg-Se soluble complex in the cells. Biogenic HgSe nanoparticles are considered the ultimate metabolite in the Hg detoxification process.

The desire to see the smallest possible objects, such as the contents of cells, reflects our intellectual curiosity and has resulted in the development of various types of microscopes. Microscopes using an X-ray source were developed after Rontgen's discovery of X-rays in 1895. Rontgen rays were first used for photography in 1896 and for observation of the structural details of biological samples in the 1900s. This use of X-rays grew considerably following the development of X-ray optics such as diffractive lenses and total-reflection mirrors in the late 1940s. X-ray microscopy theoretically has better resolution than that of visible light (400–700 nm) microscopy and has developed differently from both visible light and electron microscopy due to the penetration ability of X-rays. The third-generation synchrotron radiation facilities that produce higher electron beam energies promoted X-ray microprobes for various types of microscopies. The accompanying development of X-ray focusing systems has led to today's submicron X-ray probes, which have high enough resolution for imaging cells at the organelle level. In this review, we describe the imaging technologies using synchrotron X-ray fluorescence by means of a sub-100 nm focusing system and X-ray diffraction, which facilitates the determination of the cellular elemental distribution and structure.

We developed a detergent-free enzyme-linked immunosorbent assay (ELISA) for HIV-1 viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV), and detected soluble Vpr in ∼22% of HIV patients who were receiving combination antiretroviral therapy and were free of plasma HIV RNA. Notably, the levels of CD8-positive cell count, soluble intercellular adhesion molecule-1 (sICAM-1), and C-C motif chemokine ligand 2 (CCL2), all of which are markers of chronic inflammation in HIV patients, were higher in Vpr-positive patients than in Vpr-negative patients. Because sICAM1 and CCL2 are associated with an increased risk of HIV-associated neurocognitive disorder, we propose that an established Vpr-ELISA would be useful for monitoring the risk of HIV complications during latent HIV infection.

SALEN- and SALAN-based complexes with catalytically active metal centers are very promising small molecules to be utilized as part of antioxidant therapies. Here we discuss a modified SALAN-type molecule armed with two phosphonate groups that significantly increase its water solubility and aid to furnish mono- or dinuclear complexes with Cu2+ ions. The regulation of the SOD-mimicking (i.e., catalytic) disproportionation reaction of the superoxide radical anion (O2•−) at pH ~7.5 could be achieved by adjusting the metal-to-ligand stoichiometry as confirmed by McCord-Fridovich and pulse radiolysis tests. The higher antioxidant activity of the dicopper complex can be explained by the better access of O2•− to the copper centers and their more positive Cu(II)/Cu(I) redox potential. Simultaneously the analysis of in vitro effect on cells morphology indicates that cytotoxicity is also affected by the metal-to-ligand ratio, however, the active complex molecules do not show notable cytotoxicity that, together with the observed SOD-like activities, makes them potential candidates for antioxidant therapies.

A soft X-ray ptychography system using a Wolter mirror for the illumination optics has been developed. By taking advantage of the achromaticity of the optics, the system is capable of seamlessly imaging at half-period resolution of 50 nm with a broad photon-energy range from 250 eV to 2 keV while maintaining the focal position. Imaging a mammalian cell at various wavelengths was demonstrated, and high-resolution visualization of organelle was achieved. Stereo imaging was also performed with a long working distance of 20 mm. In combination with in-situ/operando and tomographic measurements, this system will be a powerful tool for observing biological and material targets with complex features.

Relations between structural modifiactions and SOD-like activity of four branched CuII-metallopeptides based on l-2,3-diaminopropionic acid are reported. The IC50 values (McCord–Fridovich method) illustrate that enhanced SOD-like activity is warranted by exclusive N-terminal His localization that surmounts the driving force by the CuII/I formal potential.

We have examined potential changes in the isotopic compositions of Fe, Cu and Zn (using multi-collector inductively coupled plasma-mass spectrometry) and the corresponding concentrations (using inductively coupled plasma-atomic emission spectrometry) in plasma from hematological malignancy (HM) patients and assessed their prognostic capability. Together with clinical laboratory test values, data were examined in view of a 5-years survival prediction. Plasma Cu and Zn isotope ratios and their concentrations were significantly different in HM patients compared to matched controls (P 65Cu and δ66Zn values showed significant mortality hazard ratios (HRs) in HM. The group of patients with decreased δ65Cu and increased δ66Zn values showed significantly poorer survival from the early phase (HR 3.9; P = 0.001), forming a unique cohort not identified based on laboratory test values. Well-known prognostic factors for HM, such as the creatinine level, and anemia-related values were highly correlated with the δ66Zn value (P 65Cu or δ66Zn value were similar to that based on the creatinine concentration (a well-known prognostic factor in HM), indicating that δ65Cu or δ66Zn values are useful for prognosis of HM. Variations in stable isotope ratios of essential mineral elements have thus been shown to reflect alterations in their homeostasis due to physiological changes in malignancies with higher sensitivity than concentrations do.

Cellular reprogramming is a promising technology in regenerative medicine, but most studies have been performed by using expression vectors. For future clinical applications, it is necessary to establish a system in which cell engineering can be manipulated without any risk of damaging the genome. Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11). We named the peptide “nuclear trafficking peptide” (NTP) and applied it to a protein-based artificial transcription factor (NTP-ATF), which was composed of a transcription activator-like effector and transcription domain (VP64). An NTP-ATF designed to the proximal promoter region of the microRNA-302/367 cluster efficiently induced endogenous RNA expression at an extremely low concentration (0.25 nM), and repetitive treatment of mouse embryonic fibroblasts with NTP-ATF generated induced pluripotent stem-like cells, which gave chimeric mice. Together with the observation that recombinant NTP-ATF protein did not induce any apparent cytotoxicity, we propose that NTP-ATF is a promising system for cellular reprogramming applicable to regenerative medicine.

Although mammalian target of rapamycin (mTOR) mediates a wide variety of biological functions, little information is available on the effect of mTOR on the functions of skin cells. In this study, we investigated effects of mTOR inhibition by rapamycin on ceramide synthesis in the skin of rats and human keratinocytes and its regulatory mechanisms. The phosphorylation of p70 S6 kinase, which indicates mTOR activation, was induced in the skin of rats fed a high-fat diet, but this abnormality was reversed by supplementation with rapamycin. Ceramide levels and the mRNA levels of serine palmitoyltransferase (SPT) and transforming growth factor (TGF)-β1 were suppressed in the skin of rats fed high-fat diets, but this abnormality was reversed by supplementation with rapamycin. TGF-β1-induced SPT mRNA expression was blocked by SB525334, an inhibitor of TGF-β1-induced Smad2/3 nuclear localization, in human keratinocytes. Rapamycin-induced SPT mRNA expression was blocked by an anti-TGF-β1 antibody or SB525334 in human keratinocytes. These results show that mTOR inhibition by rapamycin increases ceramide synthesis by promoting TGF-β1/Smad signaling in the skin.

Hydrocellular foam dressing (HCF) absorbs excessive wound fluid, which contains various cytokines and growth factors, and ensures a moist environment to promote wound healing. However, the molecular mechanisms underlying the wound fluid component changes induced by HCF are poorly understood. In the present study, we examined the effect of HCF on wound healing and the associated regulatory mechanisms in relation to variations in cytokine levels in the wound fluid. We created full-thickness wounds on the dorsolateral skin of rats and collected the resulting wound fluid samples. HCF was immersed in a plate containing the wound fluids. HCF was then removed and the excess wound fluid remaining in the plate was examined by cytokine array and enzyme-linked immunosorbent assay. We also used a rat model and human dermal fibroblast cultures to examine the effect of wound fluid component changes during the wound healing process. Upon treatment with HCF, leptin levels were upregulated in the wound fluid. Fibroblast proliferation was enhanced and the effect was suppressed in the presence of leptin antagonist. In our in vivo model, HCF increased wound contraction compared with film dressings and this positive effect of HCF was suppressed by addition of leptin antagonist. Our results suggest that dermal fibroblast proliferation is upregulated by HCF due to increased leptin level at the wound surface, and these effects promote wound healing. We believe that the present study contributes to furthering the understanding of the mechanisms underlying the effects of HCF-induced wound healing.

In this study, we investigated the effect of TGF-β1 on cholesterol synthesis in human keratinocytes. TGF-β1 increased the level of cholesterol and the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in human keratinocytes. These results show that TGF-β1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in human keratinocytes.

Adequate nutrition is required to maintain healthy skin integrity, and malnourished patients with poor protein diet often experience delayed wound healing. Understanding the cellular mechanisms of protein malnutrition will justify the importance of optimal protein diets in health and disease defense. Therefore in the present study, we examined the effects of changes in wound fluid composition and its function caused by protein malnutrition on wound healing. Rats were fed a control (CO; 20% protein) diet or a protein-free (PF) diet for 2 weeks; we then created full-thickness wounds on the dorsolateral skin. On day 5 after wounding, frozen sections of the wounds were created to investigate the state of granulation tissues, and wound fluid obtained from the rats was collected to examine variations in cytokine levels and its function. Wound closure was significantly delayed from day 4 until total wound closure in rats fed a PF diet. Thickness of granulation tissue, which is composed of mainly dermal fibroblasts, and Ki67 immunohistochemical staining were significantly decreased in rats fed PF diets. PF diets decreased insulin-like growth factor (IGF)-I, which promotes wound healing, and increased IGF-binding protein-1, which inhibits IGF-I bioavailability, in wound fluid. Wound fluid obtained from rats fed a PF diet suppressed dermal fibroblast proliferation. Furthermore, the wound fluid remarkably decreased the phosphorylation level of IGF-I receptor β (IGF-IR) and extracellular signal-regulated kinase (ERK)(1/2) in dermal fibroblasts. These results show that wound fluid of rats fed PF diets delays wound healing by inhibiting granulation tissue formation through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Recent technological developments have enabled the imaging of chemical elements in cells, although quantitative analyses, such as by inductively coupled plasma mass spectrometry, were developed previously. Applications allowing high-resolution imaging at the single-cell level are anticipated in cell biology and medicine, where the roles of elements, especially in relation to intracellular molecules such as proteins, nucleic acids, lipids, and sugars, are essential for understanding cellular functions. The expression of proteins and genes varies depending on cellular function, and multiple elements are likely to be associated with biological molecules in the functioning of cell proliferation, differentiation, aging, and stress responses. In this review, we describe a scanning X-ray fluorescence microscopy system, which can reliably determine the cellular distribution of multiple elements by a sub-100-nm focusing approach, together with its applications. Visualizing intracellular elements and understanding their dynamics at the single-cell level may provide great insight into their behaviors.

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Pyrrole–Imidazole (PI) polyamides bind to specific DNA sequences in the minor groove with high affinity. Specific DNA labeling by PI polyamides does not require DNA denaturation with harsh treatments of heat and formamide and has the advantages of rapid and less disruptive processing. Previously, we developed tandem hairpin PI polyamide probes (TH59 series), which label telomeres in cultured cell lines more efficiently than conventional methods, such as fluorescence in situ hybridization (FISH). Here, we demonstrate that a TH59 derivative, HPTH59-b, along with immunostaining for specifying cell types in the tissues, visualizes telomeres in mouse and human tissue sections. Quantitative measurements of telomere length with single-cell resolution suggested shorter telomeres in the proliferating cell fractions of tumor than in non-tumor tissues. Thus, PI polyamides are a promising alternative for telomere labeling in clinical research, as well as in cell biology., さまざまな組織切片の染色体テロメアの長さを3時間で検出できる方法を開発. 京都大学プレスリリース. 2016-07-06.

Chromatin DNA must be read out for various cellular functions and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug–DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.

The HIV-1 protein Vpr disrupts higher-order chromatin structure by altering histone modification and displacing important heterochromatin proteins, resulting in chromatid cohesion defects., Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1–encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.

Vpr, an accessory gene of human immunodeficiency virus type 1, encodes a virion-associated nuclear protein that plays an important role in the primary viral infection of resting macrophages. It has a variety of biological functions, including roles in a cell cycle abnormality at G(2)/M phase, apoptosis, nuclear transfer of preintegration complex, and DNA double-strand breaks (DSBs), some of which depend on its association with the chromatin of the host cells. Given that DSB signals are postulated to be a positive factor in the viral infection, understanding the mode of chromatin recruitment of Vpr is important. Here, we identified SNF2h, a chromatin-remodeling factor, as a novel binding partner of Vpr involved in its chromatin recruitment. When endogenous SNF2h protein was extensively downregulated by SNF2h small interfering RNA (siRNA), the amount of Vpr loaded on chromatin decreased to about 30% of the control level. Biochemical analysis using a mutant Vpr suggested that Vpr binds SNF2h via HFRIG (amino acids 71-75 depicted by single letters) and the Vpr mutant lacking this motif lost the activity to induce DSB-dependent signals. Consistently, Vpr-induced DSBs were attenuated by extensive downregulaion of endogenous SNF2h. Based on these data, we discuss the role of DSB and DSB signals in the viral infection.

An Amendment to this Letter has been published and is linked from the HTML version of this paper.

Visualizing the elemental distributions of cells and tissues is of growing importance in biology and medical science because such data deepen our understanding of the behavior of metal-binding proteins and ions. Elemental mapping by X-ray fluorescence analysis with a hard X-ray nanobeam is very well suited for this purpose owing to its high sensitivity and high resolution. Using this technique, samples must be prepared without artifacts that are caused by treatments such as chemical fixation and staining procedures. In many studies of elemental mapping, sample preparation is not explicitly considered. To overcome this deficiency, we developed a cryo-scanning X-ray fluorescence microscope and installed it in the second experimental hutch of BL29XUL of SPring-8. We used it to observe frozen-hydrated cells that had been fixed by a quick-freezing technique to preserve elemental data of the living state at an X-ray energy of 11.5 keV. The distributions of K, Ca, Fe, Cu and Zn were successfully visualized. The distributions of these elements (especially those of K, Ca and Fe) differed from those in cells fixed with paraformaldehyde. Copyright © 2010 John Wiley & Sons, Ltd.

A TLR4/MyD88 pathway was activated via oxidized phosphatidylcholine caused by Vpr of HIV-1. Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients’ viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-β, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.

To visualize the distributions of trace elements in biological samples such as tissues and cells at high spatial resolution, we developed a scanning x-ray fluorescence microscope (SXFM) at SPring-8, using a Kirkpatrick-Baez mirror optics that enables achromatic and highly efficient focusing. To evaluate performance regarding its application to biological samples, the SXFM was used at x-ray energy of 15 keV to observe NIH/3T3 cells in which adenosine triphosphate (ATP) synthase β (specifically localized at the mitochondria) were labeled with gold colloidal particles. Various elemental distributions were visualized at the single-cell level, including those for P, S, Cl, Ca, Fe, Cu, Zn and Au, and we obtained high-resolution elemental distribution maps by magnifying the labeled single mitochondrion. Maximum spatial resolution achieved in the experiments was sub-100 nm. Copyright © 2008 John Wiley & Sons, Ltd.

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.

Vpr, an accessory gene product of HIV-1, has been reported in the plasma of HIV-1-positive patients, and exogenous Vpr induces the reactivation of viral production from latently infected cells and the apoptosis of T cells in vitro. These observations imply that Vpr is important in AIDS development, but the clinical relevance of the findings cannot be evaluated fully because the actual plasma Vpr concentration in HIV-1-positive patients is unknown. Here we generated two monoclonal antibodies against different portions of Vpr and successfully identified Vpr as a 14-kDa protein in HIV-1-positive patients. Semiquantitative analysis using a recombinant Vpr revealed that the concentration of Vpr in patient plasma was approximately 0.7 nM (10 ng/ml). Cross-sectional analysis of 52 HIV-1-positive patients revealed that the presence of Vpr detected in 20 patients was positively correlated with HIV-1 RNA copy number (p > 0.03), but not with the number of CD4(+) T cells. This is the first report demonstrating the actual amount of Vpr in HIV-1-positive patients, and the possible linkage of Vpr and viral titers indicates that it is important to continue to carry out the sequential analysis of Vpr, especially in clinical courses of HIV-1-positive patients. The threshold of viral titers, where Vpr appears in the patients' plasma, if present, contributes to better understanding the role of Vpr in AIDS pathogenesis.

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.

Minerals are important for cellular functions, such as transcription and enzyme activity, and are also involved in the metabolism of anticancer chemotherapeutic compounds. Profiling of intracellular elements in individual cells could help in understanding the mechanism of drug resistance in tumors and possibly provide a new strategy of anticancer chemotherapy. Using a recently developed technique of scanning X-ray fluorescence microscopy (SXFM), we analyzed intracellular elements after treatment with cis-diamminedichloro-platinum(II) (CDDP), a platinum-based anticancer agent. The images obtained by SXFM (element array) revealed that the average Pt content of CDDP-resistant cells was 2.6 times less than that of sensitive cells, and the zinc content was inversely correlated with the intracellular Pt content. Data suggested that Zn-related detoxification is responsible for resistance to CDDP. Of Zn-related excretion factors, glutathione was highly correlated with the amount of Zn. The combined treatment of CDDP and a Zn(II) chelator resulted in the incorporation of thrice more Pt with the concomitant down-regulation of glutathione. We propose that the generation of an element array by SXFM opens up new avenues in cancer biology and treatment.

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

Hydrocellular foam dressing (HCF) absorbs excessive wound fluid, which contains various cytokines and growth factors, and ensures a moist environment to promote wound healing. However, the molecular mechanisms underlying the wound fluid component changes induced by HCF are poorly understood. In the present study, we examined the effect of HCF on wound healing and the associated regulatory mechanisms in relation to variations in cytokine levels in the wound fluid. We created full-thickness wounds on the dorsolateral skin of rats and collected the resulting wound fluid samples. HCF was immersed in a plate containing the wound fluids. HCF was then removed and the excess wound fluid remaining in the plate was examined by cytokine array and enzyme-linked immunosorbent assay. We also used a rat model and human dermal fibroblast cultures to examine the effect of wound fluid component changes during the wound healing process. Upon treatment with HCF, leptin levels were upregulated in the wound fluid. Fibroblast proliferation was enhanced and the effect was suppressed in the presence of leptin antagonist. In our in vivo model, HCF increased wound contraction compared with film dressings and this positive effect of HCF was suppressed by addition of leptin antagonist. Our results suggest that dermal fibroblast proliferation is upregulated by HCF due to increased leptin level at the wound surface, and these effects promote wound healing. We believe that the present study contributes to furthering the understanding of the mechanisms underlying the effects of HCF-induced wound healing.

International audience; A TAT\textlessinf\textgreater47-57\textless/inf\textgreater peptide was modified on the N-terminus by elongation with a 2,3-diaminopropionic acid residue and then by coupling of two histidine residues on its N-atoms. This branched peptide could bind to Ni under physiological conditions as a 1 : 1 complex. We demonstrated that the complex was quantitatively taken up by human fibrosarcoma cells, in contrast to Ni2+ ions. Ni localization (especially at the nuclei) was confirmed by imaging using both scanning X-ray fluorescence microscopy and Newport Green fluorescence. A competitive assay with Newport Green showed that the latter displaced the peptide ligand from the Ni-complex. Ni2+ delivered as a complex with the designed peptide induced substantially more DNA damage than when introduced as a free ion. The availability of such a construct opens up the way to investigate the importance of the nucleus as a target for the cytotoxicity, genotoxicity or carcinogenicity of Ni2+. © The Royal Society of Chemistry.

We found that exposure to room temperature (RT/21°C) causes apoptosis in HL-60 cells. Here we characterized RT-induced apoptosis in HL-60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase-1 inhibitor (YVAD-CHO) effectively blocked apoptosis, whereas the caspase-3 inhibitor (DEVD-CHO) did not. About 20% of newly obtained early passage HL-60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL-60 which responds to RT with apoptosis became dominant during passaging.

BACKGROUND HIV-positive patients have a 60-fold to 200-fold increased incidence of non-Hodgkin lymphomas, including Burkitt lymphoma, diffuse large B-cell lymphoma, and primary central nervous system lymphoma. HIV-associated lymphomas frequently have features such as extranodal involvement, decreased responses to standard chemotherapy, and high relapse rates, which indicate a poor prognosis. General pathological features do not clearly differentiate HIV-associated lymphomas from non-HIV lymphomas. METHODS To investigate the features of HIV-associated lymphomas, we performed genome-wide DNA methylation profiling of HIV and non-HIV lymphomas using Illumina GoldenGate Methylation Cancer Panel I and Illumina Infinium HumanMethylation450 BeadChip microarrays. DNA methylation profiles in HIV-associated and non-HIV lymphomas were characterized using unsupervised hierarchical clustering analyses. RESULTS The analyses of promoter regions revealed unique DNA methylation profiles in HIV-associated lymphomas, suggesting profile differences compared with non-HIV lymphomas, which implies specific gene regulation in HIV-associated lymphoma involving DNA methylation. Based on HumanMethylation450 BeadChip data, 2541 target sites were selected as differing significantly in comparisons between HIV-associated and non-HIV-associated lymphomas using Wilcoxon's rank-sum test (P

Background Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients’ blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). Results We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein β. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. Conclusions Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients’ blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases.

Approximately 42% of the human genome is composed of endogenous retroelements, and the major retroelement component, long interspersed element-1 (L1), comprises approximately 17% of the total genome. A single human cell has more than 5×105 copies of L1, 80~100 copies of which are competent for retrotransposition (RTP). Notably, L1 can induce RTP of other retroelements, such as Alu and SVA, and is believed to function as a driving force of evolution. Although L1-RTP during early embryogenesis has been highlighted in the literature, recent observations revealed that L1-RTP also occurs in somatic cells. However, little is known about how environmental factors induce L1-RTP. Here, we summarize our current understanding of the mechanism of L1-RTP in somatic cells. We have focused on the mode of L1-RTP that is dependent on the basic helix–loop–helix/per–arnt–sim (bHLH/PAS) family of transcription factors. Along with the proposed function of bHLH/PAS proteins in environmental adaptation, we discuss the functional linking of L1-RTP and bHLH/PAS proteins for environmental adaptation and evolution.

We investigated the accumulation of p53 proteins after heat stress and their association with HSP72/HSC73 using four human glioblastoma cell lines. Human glioblastoma cell lines U-87MG and A-172 exhibited no mutation in the region between the 2nd and 11th exons of the p53 gene, whereas A-7 and T98G had mutations in exon 5 and exon 7 of the p53 gene, respectively. In U-87MG and A-172, the levels of wild-type p53 protein were slightly increased by heat stress. Levels of mutant p53 protein were apparently increased by heat stress in A-7, but not in T98G. Furthermore, wild-type p53 proteins in both U-87MG and A-172 co-immunoprecipitated with anti-HSP72/HSC73 antibody and HSP72 and HSC73 in them co-immunoprecipitated with anti-p53 antibody as did the mutant p53 proteins. These findings suggest that p53 proteins accumulated by heat stress are associated with HSP72 and HSC73.

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.