Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X‐100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30–35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M_w/M_n of 2.9. ¹H NMR, ¹³C NMR, and FTIR analysis revealed the characteristic of the cis‐1,4‐polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 589–594, 2012.     

Organic acids and protein compounds causing the photoluminescence properties of natural rubber membranes and the quenching phenomena from Au nanoparticle incorporation

Natural rubber membranes were fabricated using latex from Hevea brasiliensis trees (clone RRIM 600) by casting, and controlling the time and temperature of thermal treatment. Three temperatures were used: 65, 80 and 120 °C and the corresponding annealing times of 6, 8, 10 and 12 h. The centrifugation of the latex produces the constituent phases: solid rubber (F1), serum or protein components (F2) and bottom fraction (F3). The photoluminescence properties could be correlated with organic acid components of latex. Natural rubber membranes were used as the active substrate (reducing agent) for the incorporation of colloidal Au nanoparticles synthesized by in situ reduction at different times. The intensity of photoluminescence bands assigned to the natural rubber decreases with the increase in amount of nanoparticles present on the membrane surface. It can be assumed that Au nanoparticles may be formed by reduction of the Au cation reacting with functional groups that are directly related to photoluminescence properties. However, the quenching of fluorescence may be attributed to the formation of a large amount of metal nanostructures on the natural rubber surface. Copyright © 2014 John Wiley & Sons, Ltd.     

Micro‐organisms in latex and natural rubber coagula of Hevea brasiliensis and their impact on rubber composition,structure and properties

Natural rubber, produced by coagulation of the latex from the tree Hevea brasiliensis, is an important biopolymer used in many applications for its outstanding properties. Besides polyisoprene, latex is rich in many nonisoprene components such as carbohydrates, proteins and lipids and thereby constitutes a favourable medium for the development of micro‐organisms. The fresh rubber coagula obtained by latex coagulation are not immediately processed, allowing the development of various microbial communities. The time period between tree tapping and coagula processing is called maturation, during which an evolution of the properties of the corresponding dry natural rubber occurs. This evolution is partly related to the activity of micro‐organisms and to the modification of the biochemical composition. This review synthesizes the current knowledge on microbial populations in latex and natural rubber coagula of H. brasiliensis and the changes they induce on the biochemistry and technical properties of natural rubber during maturation.     

乳胶凝集试验检测H5亚型禽流感病毒血凝素抗体的研究

利用原核表达并经过纯化的H5亚型禽流感病毒的血凝素蛋白做为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,并对偶联乳胶的蛋白量和偶联时间进行合理选择,建立H5亚型禽流感病毒血凝素蛋白乳胶凝集试验的方法。临床检测H5亚型禽流感病毒实验免疫鸡血清126份,同血凝抑制试验相比,其敏感性为87.03％,特异性为88.8％,两者的符合率为87.30%。结果证明建立的方法可用于H5亚型禽流感抗体水平监测和流行病学调查。     

Determination of 5‐hydroxytryptamine in serum by electrochemiluminescence detection with the aid of capillary electrophoresis

A rapid, sensitive and simple electrochemiluminescence method for the determination of 5‐hydroxytryptamine (5‐HT) using capillary electrophoresis was proposed. The experimental parameters, including the detection potential, the concentration of Ru(bpy)₃²⁺, the concentration and pH of phosphate buffer for separation and detection, the injection voltage and time and the separation voltage on the determination of 5‐HT, were optimized. Under the optimized conditions, the linear concentration range for 5‐HT was 3.5 × 10^‐9–5.1 × 10^‐3 mol/L, with a detection limit of 5 × 10^‐10 mol/L. The relative standard deviations (RSDs) of the ECL intensity and the migration times for six continuous injections of 1.0 µmol/L 5‐HT were 2.48% and 1.3%, respectively. The method was successfully applied to 5‐HT assay in samples of human serum in 5 min and the extraction recoveries with spiked serum samples were over 94.4%. Copyright © 2011 John Wiley & Sons, Ltd.     

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER⁺) breast cancer cell line MCF-7, but not in ER negative (ER⁻) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5′ flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER⁻ cell line MDA-MB-231 was relatively lower than that in ER⁺ MCF-7 and T47D cell lines. After the treatment with 5-aza-2′-deoxycytidine (5-aza-dC, especially in the concentration of 10 μM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.     

Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u_c) of 3.0%, and a limit of quantification (LOQ) of 1.7 μg/L when quantifying T6 as a GH-derived fragment, whereas recovery = 100.7%, SD = 2.4%, u_c = 2.5%, and LOQ = 2.7 μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.

Measurement of extracellular ATP in biological solutions is complicated by protein-binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein-bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP-stabilizing solution and endogenous plasma ATP measured. ATP-albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 micromol/L to 33.2% ATP bound at 1000 micromol/L) but independent of ATP concentration (29.3%, 10-1000 nmol/L ATP in 602 micromol/L albumin). Heating released the majority of bound ATP from albumin-containing solutions (94.8 +/- 1.7%) and plasma (97.6 +/- 5.1%). Total endogenous plasma ATP comprised 93 +/- 27 nmol/L (free) and 150 +/- 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10-1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples.     

Elevated serum kininogen in patients with Paget's disease of bone: a role in marrow stromal/preosteoblast cell proliferation

Paget's disease (PD) of bone is a chronic focal skeletal disorder characterized by excessive bone resorption followed by abundant new bone formation. Enhanced levels of IL-6, RANKL, M-CSF, and endothelin-1 have been associated with PD. In the present study, we identified increased serum levels (2 to 5-fold) of inflammatory cytokine, kininogen (KNG) in patients with PD compared to normal subjects. Treatment of pagetic bone marrow derived stromal/preosteoblast cells with recombinant KNG (25 ng/ml) for 24 h period resulted in a 5-fold increase in the levels of phospho-HSP27 and a 3-fold increase in ERK1/2 phosphorylation in these cells. However, pagetic stromal cells stimulated with KNG in the presence of ERK activation inhibitor peptide did not significantly affect the levels of phospho-HSP27. KNG increased normal and pagetic marrow stromal cell proliferation at 1.4-fold and 2.5-fold, respectively. KNG in the presence of an ERK inhibitor peptide did not stimulate pagetic marrow stromal cell proliferation. Furthermore, siRNA suppression of HSP27 expression significantly decreased KNG inhibition of etoposide-induced caspase-3 activation and apoptosis in these cells. In summary, KNG modulate bone marrow derived stromal/preosteoblast cell proliferation and suppress etoposide-induced apoptosis through ERK and HSP27 activation, respectively. These results implicate a pathophysiologic role for KNG in patients with PD.     

Prediction of efficient virological response to pegylated interferon/ribavirin combination therapy by NS5A sequences of hepatitis C virus and anti-NS5A antibodies in pre-treatment sera

A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV-1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV-1b-infected patients were treated with pegylated interferon/ ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti-NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non-EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N-terminally flanking region, which we refer to as interferon/ribavirin resistancedetermining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was signifi-cantly larger for EVR(16w) compared with Non-EVR(16w). Moreover, HCV-1b isolates with > or =5 mutations in V3 or those with > or =6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (> or =5) or IRRDR (> or =6) and the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (< or =4) or IRRDR (< or =5) together with the absence of detectable anti-NS5A antibodies could be a predictive factor for Non-EVR(16w).     

HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

HSPA5/GRP78/BiP plays an important role in cell survival or tumor progression. For these reasons, HSPA5 is an emerging therapeutic target in cancer development. Here we report that HSPA5 contributes to head and neck cancer (HNC) survival via maintenance of lysosomal activity; however, a nonthermal plasma (NTP, considered as a next-generation cancer therapy)-treated solution (NTS) inhibits HNC progression through HSPA5-dependent alteration of lysosomal activity. HSPA5 prevents NTS-induced lysosome inhibition through lysosomal-related proteins or regulation of gene expression. However, NTS-induced MUL1/MULAN/GIDE/MAPL (mitochondrial ubiquitin ligase activator of NFKB 1) leads to downregulation of HSPA5 via K48-linked ubiquitination at the lysine 446 (K446) residue. MUL1 knockdown hinders NTS-induced lysosome inhibition or cytotoxicity through the reduction of HSPA5 ubiquitination in HNC cells. While MUL1 was suppressed, HSPA5 was overexpressed in tissues of HNC patients. NTS strongly inhibited HNC progression via alterations of expression of MUL1 and HSPA5, in vivo in a xenograft model. However, NTS did not induce inhibition of tumor progression or HSPA5 reduction in MUL1 knockout (KO) HNC cells which were generated by CRISPR/Cas9 system. The data provide compelling evidence to support the idea that the regulation of the MUL1-HSPA5 axis can be a novel strategy for the treatment of HNC.     

Isolation of chlorogenic acids and their derivatives from Stemona japonica by preparative HPLC and evaluation of their anti-AIV (H5N1) activity in vitro

Two chlorogenic acids and five chlorogenic acid derivatives were simultaneously separated and purified from Stemona japonica by preparative high-performance liquid chromatography. Five of the collected compounds were over 95% pure while the other two compounds were over 90% pure. Their structures were elucidated as 3-O-feruloylquinic acid (1), 4-O-feruloylquinic acid (2), methyl 3-O-feruloylquinate (3), methyl 5-O-caffeyolquinate (4), methyl 4-O-feruloylquinate (5), ethyl 3-O-feruloylquinate (6) and the new compound ethyl 4-O-feruloylquinate (7) by UV, NMR and ESI-MS. All compounds were obtained from Stemona species for the first time, however compounds 6 and 7 are believed to be artefacts from the ethanol extraction. The anti-AIV (H5N1) activities were evaluated by Neutral Red uptake assay. Compounds 3 and 4 exerted moderate inhibitory effect against AIV (H5N1) in vitro.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.     

An Unusual Pyridine Nucleotide Accumulating in Erythrocytes: Its Identity,and Positive Correlation with Degree of Renal Failure

We have investigated an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. This nucleotide is related chemically to the NAD breakdown product, N1‐methyl‐2‐pyridone‐5‐carboxamide (Me2Py), found in high concentrations in the plasma of uraemic patients. Both Me2Py and the nucleotide accumulate to high concentrations in the blood during uraemia: our investigations of samples from renal out‐patients have provided information on a plausible link between the two compounds.