Flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) labeling to detect the cell cycle dynamics of Phaeodactylum tricornutum under light.

Cell cycle studies in plants and algae are highly dependent on reliable methods for detecting cellular DNA replication. With its short growth cycle and ease of genetic transformation, Phaeodactylum tricornutum is an important model organism for the study of pennate diatoms. Here we explored two different methods to detect the cell cycle of P. tricornutum, one using SYBR-green I to via flow cytometry, and the other using EdU labeling to observe cell cycle changes under fluorescence microscopy. Both EdU labeling fluorescence microscopy and SYBR-green I staining flow cytometry accurately indicated that the cells of P. tricornutum enter the G2/M phase after 12 h of light exposure. The results indicate that SYBR Green I was an adequate detection method for nuclear DNA quantitation in cells of P. tricornutum using a flow cytometer and without RNase A treatment. In addition, EdU can be applied to P. tricornutum to reliably detect cell proliferation. Besides, Mg-ProtoIX was able to reverse the cell cycle division inhibition of P. tricornutum and allow the nuclear DNA replication to proceed normally. Taken together, the photoperiodic division time point was clearly identified, which sheds light on the regulation of cell division mechanism in P. tricornutum.

Structural and functional analysis of the pro-domain of human cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

Comparative analysis of Xenopus tropicalis hepcidin I and hepcidin II genes.

Hepcidin is an antimicrobial peptide and an iron-regulatory hormone that is conserved in fish, amphibians, and mammalians. Here we report the genomic and biochemical characterization of two amphibian hepcidins (tHEP1 and tHEP2) from the Western clawed frog (Xenopus tropicalis). Similar to fish and mammalian hepcidins, both tHEP1 and tHEP2 genes contain three exons and two introns. The predicted mature tHEP1 and tHEP2 hepcidins are a 25 amino acid peptide and a 24 amino acid peptide, respectively. Both tHEP1 and tHEP2 are strongly expressed in the liver and kidney, with detectable expression in the heart. In addition, tHEP2 is also moderately expressed in the stomach and testis. The expression of tHEP2 (but not tHEP1) in the liver is strongly induced by iron overloading, while the expression of tHEP1 (but not tHEP2) in the liver is significantly inhibited by corticosterone. Genomic analysis of the promoter regions of these two frog hepcidin genes indicates that transcription regulation factors NF-kappaB and C/EBPbeta may be involved in hepcidin regulation by iron. Hence, X. tropicalis is a useful model for the study of molecular evolution, transcriptional regulation, and structure-activity relationships of vertebrate hepcidins.

[Increased atria expression of receptor activity-modifying proteins in heart failure patients].

OBJECTIVE: Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a calcitonin gene related peptide (CGRP) receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an adrenomedullin(ADM) receptor. As CGRP and ADM may play a beneficial role in heart failure, this study aimed at the question whether RAMPs mRNAs are changed in heart failure. METHODS: Semi-quantitative reverse transcription-PCR (RT-PCR) was used to detect and quantify the mRNAs of RAMP1 and RAMP3 in the atria of heart failing patients. RESULTS: It was found that the expressions of RAMP1, RAMP2 and RAMP3 mRNAs increased with the worsening of heart function, but the expressions of RAMP1 and RAMP2 mRNA decreased at level IV of heart failure. CONCLUSION: The above results demonstrated in the atria of heart failure patients an up-regulation of CGRP receptor by an increase of RAMP1 in association with CRLR and an up-regulation of ADM receptor by an increase of RAMP2 expression in association with CRLR, thus suggesting that CGRP and ADM receptors be playing a functional role in compensating the chronic heart failure in human.

Caspase-1 activation and mature interleukin-1ß release are uncoupled events in monocytes.

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1ß (pro-IL-1ß) and extracellular release of mature IL-1ß from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 µg/mL), and/or nigericin (Nig, 30 µmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1ß antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1ß antibodies. RESULTS: We found that pro-IL-1ß was processed to mature IL-1ß in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1ß, but not the processing of pro-IL-1ß to IL-1ß, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1ß, but not the processing of IL-1ß, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1ß processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1ß by caspase-1 and the release of mature IL-1ß from human monocytes are distinct and separable events.

Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2.

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Variation analysis of two cucumber mosaic viruses and their associated satellite RNAs from sugar beet in China.

Two cucumber mosaic virus (CMV) isolates XJ1 and XJ2 were obtained from sugar beet showing yellow mosaic symptom in Shihezi, Xinjiang Uigur municipality of China. The coat protein gene of the two CMV isolates and their associated satellite RNAs were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and were cloned and sequenced. Comparison of CP gene sequences showed that XJ1 and XJ2 have the highest sequence identity with that of CMV-Danshen (97.8%) and CMV-SD (98.7%), respectively. Two types of satellite RNAs (XJs1 and XJs2) were found to be associated with the two CMV isolates consisting of 384 nucleotides and 336 nucleotides, respectively. Sequence comparisons revealed that XJs1 and XJs2 were most closely related to CS2-sat and CS1-sat, respectively, with 98.9% and 98.5% nucleotide sequence identity. Phylogenetic analysis of nucleotide sequence and deduced amino acid sequence of coat protein gene revealed that XJ1 and XJ2 belong to subgroup IB but there exist some variation between them. Parallel analyses of nucleotide sequence of XJsl and XJs2 suggested that these two satellite RNAs probably originated from China.