Modulation of viability of live cells by focused ion‐beam exposure

Introduction of membrane‐impermeant substances into living cells is the key method to understand contemporary cellular processes by investigating cellular responses and phenotypes. Here, we performed gold ion beam exposure into live cells by using the focused ion beam implantation method, which was originally developed to precisely control semiconductor device performances. We evaluated the viability of the gold‐irradiated cells by measuring the concentration of adenosine triphosphate (ATP), which is an intracellular energy source produced in the mitochondrial membrane. The viability of the irradiated cells was found to be 20% higher than that of the unirradiated control cells. The atoms might promote the energy generating processes within the mitochondrion. Our results suggest that the viability of living cells can be modulated by accurately controlling the dopant atom numbers. Our technique may be considered as a potential tool in life and medical sciences to quantitatively elucidate the dose‐dependent effects of dopants. Biotechnol. Bioeng. 2011; 108:222–225. © 2010 Wiley Periodicals, Inc.     

Depressive symptoms of female nursing staff working in stressful environments and their association with serum creatine kinase and lactate dehydrogenase – a preliminary study

Background

The activity of creatine kinase (CK) in serum has recently been reported to be potentially associated with several types of depression. The aim of this study is to evaluate whether serum enzymes, including CK, vary even in a healthy population with depressive symptoms caused by work-related stress. We gave questionnaires and blood examinations to 93 healthy female nursing home workers and did an enzyme-linked immunosorbent assay for the quantitative detection of CK isozyme muscle-type M chain (CK-MM) in serum.

Findings

Depressive symptoms were determined using the Center for Epidemiologic Studies Depression (CES-D) scale and compared with the results of the blood examination and serum CK-MM levels. The CES-D results showed significant negative correlations with total CK and lactate dehydrogenase (LDH) activities and CK-MM level (r?=?-0.29, p?=?0.0062; r?=?-0.29, p?=?0.0065; r?=?-0.33, p?=?0.0016, respectively).

Conclusions

Total CK and LDH activities and serum CK-MM level appear to be associated with the depressive symptoms of healthy nurses working in stressful environments, although the significance level was relatively low. The simultaneous detection of serum CK and LDH activities or serum CK-MM level and LDH activity may be useful as an indicator of depressive symptoms, at least for female nursing staff with work-related stress.

     

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.     

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Locus of dominant hairless gene (Ht) causing abnormal hair and keratinization maps to rat chromosome 10.

The rat dominant hairless gene (Ht) of the WBN/Ila-Ht rat causes atrichosis in Ht/Ht and hypotrichosis in Ht/+. Furthermore the Ht/Ht shows signs of abnormal keratinization and almost all of the Ht/Ht die in an immature stage before weaning in the conventional environment. Ht/+ was affected by dermatitis caused by Staphylococcus aureus, suggesting that the gene Ht might involve defense mechanisms against infection. In this study, we performed the linkage analysis of the gene Ht by outcross with the Brown Norway rat in the SPF environment. Ninety-six backcross progeny of (BN x WBN/Ila-Ht/Ht) F1 x WBN/Ila-Ht/Ht were typed with microsatellite markers and the gene Ht was mapped on chromosome 10 between Asgr1 and Nos2 within the map distance of 6.2 cM.     

Local adaptation and host race formation of a gall-forming aphid in relation to environmental heterogeneity

Summary The process of host race formation in the aphid Tetraneura yezoensis is examined in relation to its population structure. T. yezoensis induces pouch galls on new leaves of Ulmus davidiana and U. laciniata. Its populations on the two host species are often sympatric. Fundatrices found on one elm species, when reciprocally transplanted to the other, suffered greatly reduced average fitness. This shows that aphid populations associated with the two elm species are genetically differentiated in physiological traits. Individual trees of each elm species showed large differences in susceptibility to gall formation and in bud burst time, and such between-tree variations were consistent over years. Overwintered eggs taken in early spring from four trees (two from each species) were incubated under the same temperature conditions. The average hatching time differed significantly even between populations from conspecific trees, and the sequence of egg hatching paralleled that of the leafing of those four trees. This between-tree difference in hatching time was consistent over years and was found to be genetic, showing that gene flow between aphid populations on separate trees is often restricted. The heterogeneity in host traits may have promoted the evolution of philopatry in this aphid. Of the fundatrices that hatched on a tree of one elm species, a few precent were preadapted to gall formation on the other elm species. This suggests that the formation of a new host race proceeds parapatrically under disruptive selection and at a low level of gene flow. Evidence was actually obtained that a small fraction of Tetraneura alates are passively transported and land on non-host plants.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.