Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.     

Chemical reactivity of the carbon-centered free radicals and ferrous iron in coals: Role of bioavailable Fe2+ in coal workers' pneumoconiosis

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe²⁺ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe²⁺ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe²⁺ with O₂ in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe²⁺, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe²⁺-induced oxidative stress and eventual CWP development.     

Inhibition of 2-P-mercaptophenyl-1,4-naphthoquinone on human platelet function.

As widely assumed, platelets and coagulation system heavily influence the pathogenesis and progression of cardiovascular diseases. Some 1,4-naphthoquinone derivatives, such as vitamin K3, have been reported to increase the synthesis of coagulation proteins. In this study, we examine how 2-p-mercaptophenyl -1,4-naphthoquinone (NTP), a newly synthesized 1,4-naphthoquinone derivative, affects the platelet function in humans. A tapered parallel plate chamber which provided a range of shear stress covering the entire physiological range in human circulation is used to assess platelet adhesiveness on fibrinogen coated-surface. In addition, platelet aggregation and thromboxane B2 (TXB2) production by inducers are evaluated by the turbidimetric method and enzyme immunoassay kit, respectively. Moreover, platelets [Ca2+]i are measured using a dual-wavelength fluorescence spectrophotometer. Analysis results indicate that 1) NTP decreases the percentages of attached platelets at the locations in various shear stresses and the levels of platelet adhesiveness, denoted as the slope; 2) NTP can inhibit the platelet aggregation by ADP (2 microM) and collagen (25 microg/ml), and the IC50 are: 0.32 and 26.83 microg/ml, respectively; and 3) NTP markedly inhibits TXB2 formation and platelet [Ca2+]i elevation caused by ADP and collagen. Therefore, we conclude that NTP may inhibit platelet adhesiveness on fibrinogen coated-surface, aggregability, [Ca2+]i, and thromboxane production, and that it may be used as an antiplatelet agent.     

Role of exonucleolytic degradation in group I intron homing in phage T4

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).     

流行性出血热地鼠肾细胞灭活疫苗人体试用细胞免疫反应观察

观察了20位志愿者在接种试验性流行性出血热(EHF)地鼠肾细胞(GHKC)双价灭活疫苗后的细胞免疫反应,并以其体液免疫反应作对照观察。用淋巴细胞转化试验测定细胞免疫水平。结果首针疫苗接种后42天和56天,特异性刺激指数(SSI)和非特异性刺激指数(NSI)均较免疫前显著增高(P_(SSI)<0.001;P_(NSI)<0.02),免疫后SSI累计阳转率为100％,NSI累计阳转率为60％,免疫后6个月二者均降低至正常水平。免疫后56天测定抗体,荧光抗体阳转率为100％;微量感染性中和试验表明,针对家鼠型病毒L99株中和抗体阳转率为95％,而针对野鼠型病毒JR株阳转率为65％。     

Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish

Mutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and non-photoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV- and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.     

Long-term Therapeutic Effects of Extracorporeal Shock Wave-Assisted Melatonin Therapy on Mononeuropathic Pain in Rats

We evaluated the ability of extracorporeal shock wave (ECSW)-assisted melatonin (Mel) therapy to offer an additional benefit for alleviating the neuropathic pain (NP) in rats. Left sciatic nerve was subjected to chronic constriction injury (CCI) to induce NP. Animals (n?=?30) were randomized into group 1 (sham-operated control), group 2 (CCI only), group 3 (CCI?+?ECSW), group 4 (CCI?+?Mel) and group 5 (CCI?+?ECSW?+?Mel). By days 15, 22 and 29 after CCI, the thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but they showed no difference between the later two groups (all p?<?0.0001). The protein expressions of inflammatory (TNF-α, NF-κB, MMP-9, IL-1ß), oxidative-stress (NOXs-1, -2, -4, oxidized protein), apoptotic (cleaved-caspase3, cleaved-PARP), DNA/mitochondrial-damaged (γ-H2AX/cytosolic-cytochrome C), microglia/astrocyte activation (ox42/GFAP), and MAPKs [phosphorylated (p)-p38, p-JNK, p-ERK] biomarkers in dorsal root ganglia neurons (DRGs) and in spinal dorsal horn were exhibited an opposite pattern of TPWL among the five groups (all p?<?0.0001). Additionally, protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve exhibited an identical pattern to inflammation among the five groups (all p?<?0.0001). The numbers of cellular expressions of MAPKs (p-ERK1/2+/peripherin?+?cells, p-ERK1/2+/NF200?+?cells and p-JNK+/peripherin?+?cells, p-JNK+/NF200?+?cells) and voltage-gated sodium channels (Nav.1.8+/peripherin?+?cells, Nav.1.8+/NF200?+?cells, Nav.1.9+/peripherin?+?cells, Nav.1.9+/NF200?+?cells) in small and large DRGs displayed an identical pattern to inflammation among the five groups (all p?<?0.0001). In conclusion, the synergistic effect of combined ECSW-Mel therapy is superior to either one alone for long-term improvement of mononeuropathic pain-induced by CCI in rats.

     

Long-term exposure to ambient air pollution and mortality due to cardiovascular disease and cerebrovascular disease in Shenyang, China

Background

The relationship between ambient air pollution exposure and mortality of cardiovascular and cerebrovascular diseases in human is controversial, and there is little information about how exposures to ambient air pollution contribution to the mortality of cardiovascular and cerebrovascular diseases among Chinese. The aim of the present study was to examine whether exposure to ambient-air pollution increases the risk for cardiovascular and cerebrovascular disease.

Methodology/Principal Findings

We conducted a retrospective cohort study among humans to examine the association between compound-air pollutants [particulate matter <10 µm in aerodynamic diameter (PM₁₀), sulfur dioxide (SO₂) and nitrogen dioxide (NO₂)] and mortality in Shenyang, China, using 12 years of data (1998–2009). Also, stratified analysis by sex, age, education, and income was conducted for cardiovascular and cerebrovascular mortality. The results showed that an increase of 10 µg/m³ in a year average concentration of PM₁₀ corresponds to 55% increase in the risk of a death cardiovascular disease (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.51 to 1.60) and 49% increase in cerebrovascular disease (HR, 1.49; 95% CI, 1.45 to 1.53), respectively. The corresponding figures of adjusted HR (95%CI) for a 10 µg/m³ increase in NO₂ was 2.46 (2.31 to 2.63) for cardiovascular mortality and 2.44 (2.27 to 2.62) for cerebrovascular mortality, respectively. The effects of air pollution were more evident in female that in male, and nonsmokers and residents with BMI<18.5 were more vulnerable to outdoor air pollution.

Conclusion/Significance

Long-term exposure to ambient air pollution is associated with the death of cardiovascular and cerebrovascular diseases among Chinese populations.