Comparison and Risk Assessment for Trace Heavy Metals in Raw Pu-erh Tea with Different Storage Years.

This research conducted an exploration of the content of microelements (As, Cr, Cd, Pb, Cu, Zn, Mn, and Hg) in raw Pu-erh tea with different storage years. The contents of As, Cr, Cd, Pb, Cu, Zn, Mn, and Hg were 0.14, 0.82, 0.02, 0.52, 14.59, 33.51, 564.02, and 0.01 µg/g, respectively, and were all less than the national standard limit values in China. The target hazard quotients (THQs) of each heavy metal were all lower than 1, and the value of combined risk hazard index (HI) of all to adults was 0.221, which presents no health risk when consumed properly by adults of the raw Pu-erh tea infusions. Interestingly, there was no significant correlation between the heavy metal element (As, Cr, Cd, Pb, Cu, Zn, Mn, and Hg) contents and the THQ values of raw Pu-erh tea samples and storage years; the correlation coefficients (R2) range from 0.01 to 0.33 and from 0.01 to 0.57, respectively. The result showed that the storage years showed no effect on the exposure risk of heavy metals; the heavy metal elements in tea samples come from the atmosphere and soil.

Expression of interleukin-15 and its receptor on the surface of stimulated human umbilical vein endothelial cells.

Human interleukin-15 (hIL-15) is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) to induce the production of human interleukin-15 (hIL-15) and IL-15 receptor (IL-15Ralpha) by human umbilical vein endothelial cells (HUVECs). The data are summarized as follows: 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-gamma and TNF-alpha. 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting (FACS), and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Ralpha was detected on the surface of HUVECs by FACS after IFN-gamma and TNF-alpha stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Ralpha was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-gamma and TNF-alpha play an important role in regulating the expression of IL-15 and IL-15Ralpha on the surface of HUVECs.

Arabidopsis Histone Methyltransferase SUVH5 Is a Positive Regulator of Light-Mediated Seed Germination.

Plant lifecycle starts from seed germination, which is regulated by various environmental cues and endogenous hormones. Light promotes seed germination mainly by phytochrome B (PHYB) during the initial phase of imbibition, which involves genome-wide light-responsive transcription changes. Recent studies indicated an involvement of multiple epigenetic factors in the control of seed germination. However, few studies have been reported about the role of a histone methyltransferase in light-mediated seed germination process. Here, we identified SUVH5, a histone H3 lysine 9 methyltransferase, as a positive regulator in light-mediated seed germination in Arabidopsis. Loss of function of SUVH5 leads to decreased PHYB-dependent seed germination. RNA-sequencing analysis displayed that SUVH5 regulates 24.6% of light-responsive transcriptome in imbibed seeds, which mainly related to hormonal signaling pathways and developmental processes. Furthermore, SUVH5 represses the transcription of ABA biosynthesis and signal transduction-related genes, as well as a family of DELAY OF GERMINATION (DOG) genes via dimethylation of histone H3 at lysine 9 (H3K9me2) in imbibed seeds. Taken together, our findings revealed that SUVH5 is a novel positive regulator of light-mediated seed germination in Arabidopsis.

The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors.

The solution structure of the potent 95 residue anti-HIV protein scytovirin has been determined and two carbohydrate-binding sites have been identified. This unique protein, containing five structurally important disulfide bonds, demonstrates a novel fold with no elements of extended regular secondary structure. Scytovirin contains two 39 residue sequence repeats, differing in only three amino acid residues, and each repeat has primary sequence similarity to chitin binding proteins. Both sequence repeats form similarly structured domains, with the exception of one region. The result is two carbohydrate-binding sites with substantially different affinities. The unusual fold clusters aromatic residues in both sites, suggesting a binding mechanism similar to other known hevein-like carbohydrate-binding proteins but differing in carbohydrate specificity. Scytovirin, originally isolated from the cyanobacterium Scytonema varium, holds potential as an HIV entry inhibitor for both therapeutic and prophylactic anti-HIV applications. The high-resolution structural studies reported are an important initial step in unlocking the therapeutic potential of scytovirin.

Modulation of interleukin-15-induced suppression of human neutrophil apoptosis by TNFalpha.

Human interleukin-15 (IL-15) is a proinflammatory cytokine to suppress neutrophil apoptosis, which is a potential therapeutic agent. The modulatory effect of TNFalpha was investigated in IL-15-induced suppression of human neutrophil apoptosis. TNFalpha was shown to reverse the ability of IL-15 to delay neutrophil apoptosis within certain time course. Moreover, this reverse effect by TNFalpha might be associated with a reduction of the expression of the anti-apoptotic Bcl-Xl protein detected by Western blotting. It is concluded that TNFalpha can be used to modulate IL-15-induced suppression of neutrophil apoptosis within certain time course.

Potent anti-HIV activity of scytovirin domain 1 peptide.

Scytovirin (SVN) is a novel anti-HIV protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. SVN contains two apparent domains, one comprising amino acids 1-48 and the second stretching from amino acids 49 to 95. These two domains display significant homology to each other and a similar pattern of disulfide bonds. Two DNA constructs encoding scytovirin 1-48 (Cys7Ser) (SD1) and 49-95 (Cys55Ser) (SD2) were constructed, and expressed in E. coli, with thioredoxin fused to their N-terminus. Purified recombinant products were tested for binding activities with the HIV surface envelope glycoproteins gp120 and gp41. Whole cell anti-HIV data showed that SD1 had similar anti-HIV activity to the full-length SVN, whereas SD2 had significantly less anti-HIV activity. Further deletion mutants of the SD1 domain (SVN(3-45)Cys7Ser, SVN(6-45)Cys7Ser, SVN(11-45)Cys7Ser) showed that the N-terminal residues are necessary for full anti-HIV activity of SD1 and that an eight amino acid deletion from the C-terminus (SVN(1-40)Cys7Ser) had a significant effect, decreasing the anti-HIV activity of SD1 by approximately five-fold.

Cloning and expression of rabbit interleukin-15.

In order to understand the inflammatory mechanisms related to rabbit interleukin-15 (RIL-15), we cloned and expressed RIL-15 cDNA gene. A cDNA encoding RIL-15 was cloned from heart mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using hIL-15 primers. The RIL-15 cDNA contains an open reading frame (ORF) of 162 amino acids (aa) with a 48 aa leader sequence. The predicted molecular weight of the encoded protein (12.5 kDa) matched the size of recombinant IL-15 on Western blotting in an Escherichia coli (pET32a) expression system. Amino acid and nucleotide sequence analyses of RIL-15 revealed 82.7% and 87% homology with human IL-15 (hIL-15), respectively. RIL-15 is similar to the hIL-15 (hIL-15) in that it contains seven cysteine residues. RT-PCR showed that IL-15 is expressed in many tissues in the rabbit, including heart, spleen, lung, liver, muscle and kidney. Expressed and purified recombinant RIL-15, in the absence of the 48 aa leader sequence, stimulated the proliferation of cells of the mouse T cell line, CTLL-2, and its activity is comparable to hIL-15. Western blotting demonstrated that recombinant RIL-15 can be recognized by anti-IL-15 neutralization antibody. Western blotting also confirmed that IL-15 is present in many tissues including heart, spleen, lung, liver, muscle and kidney.

Isolation, Purification and Characterization of a Group of Novel Small Molecular Ribosome Inactivating Protein -- LuffinS from Seeds of Luffa cylindrica.

A group of novel RIPs--LuffinS(1), LuffinS(2), LuffinS(3) (MW about 8 kD) were purified by ammonia sulfate precipitation, CM-52 chromatography, HRLC size chromatography and Mono S FPLC. LuffinS(1), LuffinS(2) and LuffinS(3) have similar weight of about 8kD, and their N-terminal amino acid is Ala, Pro and Thr respectively. The N-terminal nine amino acid sequence of LuffinS(2) was determined as Pro-Arg-Arg-Gly-Gln-Glu-Ala-Phe-Asp. The reaction mechanism of LuffinSs is the same as that of TCS, which is RNA N glycosidases. LuffinSs are more toxic than TCS, with IC(50) of 1.3x10(-11), 1.0x10(-10) and 6.3x10(-11) mol/L respectively in a cell-free protein synthesis system. It is promising that LuffinSs may be used as the efficient toxin moiety of immunotoxins.

In vitro Inhibition of Human Melanoma Cells by Immunotoxin Luffin B-Ng76.

Luffin B, a plan single-chain ribosome inactivating protein, was purified from seeds of Luffa cylindrica by Blue Sepharose CL-6B affinity chromatography. An immunotoxin was constructed with luffin B and Ng76, a monoclonal antibody to human melanoma cell M(21). Luffin B-Ng76 showed 4 000-fold more cytotoxic to target melanoma cells than free luffin B. The IC(50) of luffin B-Ng76 for M(21) cells and non-target HeLa cells was 2.5x10(-11) mol/L and 3.0x10(-8) mol/L, respectively. The results suggest that luffin B is a new potent immunotoxin effector.

Overexpression and purification of scytovirin, a potent, novel anti-HIV protein from the cultured cyanobacterium Scytonema varium.

Scytovirin (SVN) is a novel anti-human immunodeficiency virus (HIV) protein isolated from aqueous extracts of the cultured cyanobacterium Scytonema varium. The protein consists of a single 95-amino acid chain with significant internal sequence duplication and 10 cysteines forming five intrachain disulfide bonds. A synthetic gene that encodes scytovirin was constructed, and expressed in Escherichia coli, with thioredoxin (TRX) fused to its N-terminus (TRX-SVN). Most of the expressed protein was in soluble form, which was purified by a polyhistidine tag affinity purification step. SVN was then cleaved from TRX with enterokinase and separated from the TRX partner by C18 reversed-phase HPLC. This production method has proven superior to earlier synthetic attempts and recombinant procedures using a standard expression system. The current system resulted in yields of 5-10mg/L of purified SVN for structural studies and for preclinical development of SVN as a topical microbicide for HIV prophylaxis.

Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library.

Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties.