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.
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.
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-κB and C/EBPβ 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.
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.
Nitella fuiana Lan & Qing sp. nov., a special kind of charophyte from Mingshan, Sichuan, China, was described in this study. The systematic position of the new species was assessed based on ...morphological and molecular analysis. The results of morphology comparison showed that the charophyte possessed stipulodes and 10-cell coronula. Nuclear 18S rDNA and plastid rbcL sequences analysis revealed that N. fuiana was a single clade as clustered with other species of Nitella. Combined with the morphological and molecular analysis data, our study strongly supported that N. fuiana was a new species of Nitella. This is the first report of Nitelleae species with stipulodes, which increased the morphological diversity of charophytes. This study also reminds us to pay close attention to the characteristics of stipulodes for further research on charophytes.
To evaluate the sterilization effect of electron beam irradiation on Tibetan medicine Bawei Chenxiang powder and the influence on the active ingredients, medicine samples were irradiated with ...electron beam at doses of 0 kGy, 5 kGy, 7 kGy, 10 kGy, and 60Co γ at 7 kGy. The microbial and active substance costunolide levels were measured within 7 d after irradiation and after accelerated storage at 49 ℃ for 3 months, respectively. The results showed that electron beam and 60Co γ irradiation significantly reduced the total number of microorganisms in Bawei Chenxiang powder. Irradiation by electron beams at 5 kGy and 10 kGy reduced the total number of microorganisms in Bawei Chenxiang powder to the maximum requirement suggested by the Chinese Pharmacopoeia without significantly decreasing the amount of active ingredient. In conclusion, electron beam irradiation can effectively improve the microbial index of Bawei Chenxing powder, and 5 kGy is the optimal processing dose for this purpose.
Abstract
We have previously shown that human defensins can block the release of IL-1beta from LPS-primed human monocytes stimulated with ATP. The objective of this study is to determine whether ...defensins can inhibit the release IL-1beta and IL-18 from LPS-primed human monocytes stimulated with other inflammasome activators. LPS-activated (20 ng/ml, 2 h), 35S-methionine-labeled (1 h) human monocytes were treated with human defensin HD-5 in the presence or absence of ATP, monosodium urate (MSU), nigericin (Nig) for 90 min. Media and cell-associated fractions were harvested separately. IL-1beta was recovered from each by immunoprecipitation and caspase-1 was examined by western blot analysis. Mature IL-1beta and active caspase-1 were detected in the cytosol fraction from cells treated with HD-5. Thus, HD-5 blocked the release of mature IL-1beta from cells treated with ATP, MSU, or Nig. However, HD-5 did not inhibit ATP-, MSU-, or Nig-induced caspase-1 activation and processing of IL-1beta. In addition, HD-5 also blocked the release of IL-18 from LPS-primed human monocytes stimulated with ATP. To our knowledge, HD-5 is the first known endogenous inhibitor of IL-1beta and IL-18 release. These studies suggest that processing and release of IL-1beta are independent processes.
We have previously shown that human alpha‐defensins can block the release of IL‐1beta from LPS‐activated human monocytes. The objective of this study is to use defensins as tools to determine the ...molecular mechanism by which IL‐1beta is processed and secreted from human monocytes. LPS‐activated (20 ng/ml, 2 h), 35S‐methionine‐labeled (1 h) human monocytes were treated with human alpha‐defensin HD‐5 in the presence or absence of ATP, staphylococcal alpha‐toxin (SAT), or nigericin (Nig) for 3 h. Media and cell–associated fractions were harvested separately. IL‐1beta was recovered from each by immunoprecipitation and caspase‐1 was examined by western blot analysis. It was found that HD‐5 blocked the release of mature IL‐1beta from cells treated with ATP or Nig, while HD‐5 did not inhibit ATP‐ or Nig‐induced caspase‐1 activation and externalization. However, SAT‐induced caspase‐1 activation was blocked by HD‐5 and cytosolic mature IL‐1beta was not detected in cells treated with SAT and HD‐5. To our knowledge, HD‐5 is the first known endogenous inhibitor of IL‐1beta release. This study suggest that although both IL‐1beta and caspase‐1 are detected in the extracellular milieu after ATP stimulation, mature IL‐1beta and its converting enzyme caspase‐1 are secreted via different pathways.
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.
PDF
