Effect of Shaoyaotang on Expressions of CD14, FADD and Caspase-8 in Colonic Tissues of Rats with Large Intestinal Damp-heat Syndrome of Ulcerative Colitis / 中国实验方剂学杂志

Objective:To observe the effect of Shaoyaotang on the contents of cell adhesion molecule-1 （ICAM-1） and transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in serum of large intestine damp-heat syndrome of ulcerative colitis （UC） in rats， and the gene and protein expressions of leukocyte differentiation antigen14 （CD14）， Fas-related death domain protein （FADD） and cysteinyl aspartate specific protease-8 （Caspase-8） in the focal colon tissue. Method:A total of 80 SPF Wistar rats were randomly divided into the blank group （<italic>n</italic>=10） and modeling group （<italic>n</italic>=70）. The large intestine damp-heat syndrome of UC rats was replicated by the combination of disease and syndrome， which was high-fat， high-sugar and spicy diets combined with 2， 4-dinitrobenzene sulfonic acid （DNBS） and ethanol. After successful modeling， the modeled groups were divided into model group， sulfasalazine （SASP）control group， and low， medium and high-dose Shaoyaotang groups by the method of random number table， with14 rats in each group. Low， medium and high doses of Sulfasalazine 0.2 g·kg^-1·d^-1 and Shaoyaotang （6， 12， 24 g·kg^-1·d^-1）were given by gavage. The blank group and the model group were given equal volume of normal saline for 21 days. The contents of serum ICAM-1 and TGF-<italic>β</italic>₁ were detected by enzyme-linked immunosorbent assay （ELISA）， the expressions of CD14， FADD and Caspase-8 mRNA in colon tissues were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）， and the expressions of CD14， FADD and Caspase-8 protein in colon tissues were detected by Western blot. Result:Compared with the blank group， the serum ICAM-1 level in the model group were significantly increased， whereas the content of TGF-<italic>β</italic>₁ were significantly decreased （<italic>P</italic><0.05）. The relative expression levels of CD14， FADD， Caspase-8 mRNA and protein were significantly increased （<italic>P</italic><0.05）. Compared with the model group， the content of ICAM-1 in the serum of the rats in the medium， high-dose Shaoyaotang groups and the SASP group were significantly decreased， while the content of TGF-<italic>β</italic>₁ in the serum of the rats in the low， medium， high-dose Shaoyaotang groups and the SASP group were significantly increased （<italic>P</italic><0.05）. The expression levels of CD14， FADD， Caspase-8 mRNA and protein in each intervention group were significantly decreased （<italic>P</italic><0.05）， especially in the high-dose Shaoyaotang group and the SASP group. Conclusion:Shaoyaotang has a certain intervention effect on UC rats with large intestine damp-heat syndrome， and its mechanism may be related to the inhibition of CD14， FADD and Caspase-8 genes and proteins expression.

Expressions of IL-1α, IL-1β and IL-13 mRNA in Lung Tissue and Serum of Rats Drown in Fresh Water / 法医学杂志

Objective To detect the changes of (interleukin, IL) -1α, IL-1β and IL-13 mRNA in lung tissue and serum of drown rats, and to explore the potential value for the diagnosis of drowning in forensic practice. Methods Eighteen SD rats were randomly divided into drowning group, blank control group and myocardial infarction group (as control group). The serum of right ventricular, the inferior lobe of right lung and the myocardium were taken from the rats in different groups. The expressions of IL-1α, IL-1β and IL-13 mRNA in the lung tissue and the serum of right ventricular were detected by TaqMan probe method. Results The expression differences of IL-1α, IL-1β and IL-13 mRNA in lung tissue between drowning group and blank control group, myocardial infarction group were not statistically significant (P>0.05). The expression of IL-1β and IL-13 mRNA in serum of right ventricular increased (P<0.05). The expression differences of IL-1α, IL-1β and IL-13 mRNA in serum between blank con-trol group and myocardial infarction group were not statistically significant (P>0.05). Conclusion The changes of cytokines IL-1β and IL-13 mRNA in the serum of right ventricular of drown rats are statis-tical significance, which are highly correlated with drowning.

The emerging roles of the DDX41 protein in immunity and diseases

RNA helicases are involved in almost every aspect of RNA, from transcription to RNA decay. DExD/H-box helicases comprise the largest SF2 helicase superfamily, which are characterized by two conserved RecA-like domains. In recent years, an increasing number of unexpected functions of these proteins have been discovered. They play important roles not only in innate immune response but also in diseases like cancers and chronic hepatitis C. In this review, we summarize the recent literatures on one member of the SF2 superfamily, the DEAD-box protein DDX41. After bacterial or viral infection, DNA or cyclic-di-GMP is released to cells. After phosphorylation of Tyr414 by BTK kinase, DDX41 will act as a sensor to recognize the invaders, followed by induction of type I interferons (IFN). After the immune response, DDX41 is degraded by the E3 ligase TRIM21, using Lys9 and Lys115 of DDX41 as the ubiquitination sites. Besides the roles in innate immunity, DDX41 is also related to diseases. An increasing number of both inherited and acquired mutations in DDX41 gene are identified from myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML) patients. The review focuses on DDX41, as well as its homolog Abstrakt in Drosophila, which is important for survival at all stages throughout the life cycle of the fly.

In vitro expression and analysis of the 826 human G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.

Conventional Tumor Markers in Cerebralspinal Fluid in Patients with Elevated Serum Tumor Markers and without Central Nervous System Malignant Diseases / 中国医学科学院学报

Objective To explore the diffusion pattern of tumor markers (TM) from serum to cerebrospinal fluid (CSF) via the blood-brain barrier in patients with elevated serum tumor markers (TM).Methods Inpatients receiving lumbar puncture during hospitalization in our center from January 1, 2013 to December 31, 2015 were divided into study group (n=181) and control group (n=251). The study group consisted of patients with elevated serum TMs but without malignant central nervous system diseases. The control group consisted of patients with normal serum TM levels and without malignant diseases. TMs measured in the study group included elevated serum alpha-fetoprotein (AFP) (n=0), carcinoembryonic antigen (CEA) (n=26), carcinomic antigen(CA)125 (n=39), CA15- 3 (n=3),CA19- 9 (n=19), CA724 (n=47), CYFRA21- 1 (n=49), and SCC (n=17).Levels of TMs in the CSF of study group was compared with that of control group.Results Median CEA (U=0.00,P=0.00),CA19- 9 (U=0.00,P=0.00),CA15- 3 (U=0.00,P=0.04),SCC (U=0.00,P=0.00),CA125 (U=0.00,P=0.00),CA72- 4 (U=3.00,P=0.00)),and CYFRA21- 1 (U=0.00,P=0.00) in CSF were significantly lower than the corresponding serum TM levels in the study group.There was no significant difference between study group and control group for the CSF level of CEA (U=3091.00,P=0.18),CA19- 9 (U=1897.00,P=0.14), CA15- 3 (U=373.50,P=0.91)and SCC (U=1925.50,P=0.76). CSF CA125 (U=2188.00,P=0.00) and CA724 (U=1279.00,P=0.00) levels in the study group were lower than those in control group. CSF level of CYFRA21- 1 (U=1826.50,P=0.00) in study group was higher than that in control group;however, it was still lower than the upper limit of reference value. Conclusion In patients with elevated serum CEA, CA19- 9, CA15- 3, SCC, CA125, and CA72- 4 levels, transblood-brain-barrier diffusion of TMs from serum to CSF is highly unlikely.

Mechanism of the Rpn13-induced activation of Uch37

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body's defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING(-/-) mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.

Structural biology study of human TNF receptor associated factor 4 TRAF domain

TRAF4 is a unique member of TRAF family, which is essential for innate immune response, nervous system and other systems. In addition to be an adaptor protein, TRAF4 was identified as a regulator protein in recent studies. We have determined the crystal structure of TRAF domain of TRAF4 (residues 292-466) at 2.60 Å resolution by X-ray crystallography method. The trimericly assembled TRAF4 resembles a mushroom shape, containing a super helical "stalk" which is made of three right-handed intertwined α helixes and a C-terminal "cap", which is divided at residue L302 as a boundary. Similar to other TRAFs, both intermolecular hydrophobic interaction in super helical "stalk" and hydrogen bonds in "cap" regions contribute directly to the formation of TRAF4 trimer. However, differing from other TRAFs, there is an additional flexible loop (residues 421-426), which contains a previously identified phosphorylated site S426 exposing on the surface. This S426 was reported to be phosphorylated by IKKα which is the pre-requisite for TRAF4-NOD2 complex formation and thus to inhibit NOD2-induced NF-κB activation. Therefore, the crystal structure of TRAF4-TRAF is valuable for understanding its molecular basis for its special function and provides structural information for further studies.

Effect of human placental extract on proliferation of human umbilical cord blood CD34(+) cells in vitro / 中国实验血液学杂志

The study was aimed to investigate the hematopoietic function of placenta tissue and clarify the effect of human placental chorionic tissue in different periods on proliferation of hematopoietic stem cells in vitro, in order to further understand the changes of the hematopoietic function of placenta with the time prolonging. The experiments were divided into four groups: early placenta (group B), mid-term placenta (group C), full-term placenta (group D), and blank group (group A). The hematopoietic stem cells were amplified in co-culture way, and the colony formation ability after the expansion was observed. The results showed that compared to initial concentration, the CD34(+) cells cultured with full-term placenta were amplified by (2.60 ± 0.20) times, which was significantly higher than those CD34(+) cells cultured with mid-term placenta (1.74 ± 0.24) and early placenta (1.14 ± 0.12), but that in blank group was reduced without amplification. After culture for 14 days, the colony number of group C and group D were significantly higher than that of group A and group B. Among them the number of CFU-GM, CFU-GEMM, BFU-E of group C all were a little higher than that of group D. It is concluded that human placental extract in different period without any exogenous cell factors all can support the proliferation of hematopoietic stem cells, this ability is getting stronger with time increasing. The colony formation ability of the amplified cells shows weakened after the first increase, this colony formation ability of the amplified cells in group C is strongest, slightly stronger than that of group D.

Protein-protein complexation in bioluminescence

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.

Crystal structure of a novel non-Pfam protein PF2046 solved using low resolution B-factor sharpening and multi-crystal averaging methods

Sometimes crystals cannot diffract X-rays beyond 3.0 Å resolution due to the intrinsic flexibility associated with the protein. Low resolution diffraction data not only pose a challenge to structure determination, but also hamper interpretation of mechanistic details. Crystals of a 25.6 kDa non-Pfam, hypothetical protein, PF2046, diffracted X-rays to 3.38 Å resolution. A combination of Se-Met derived heavy atom positions with multiple cycles of B-factor sharpening, multi-crystal averaging, restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5 (R(free)= 24.7). The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers. Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.