A novel putative betapartitivirus isolated from the plant-pathogenic fungus Rhizoctonia solani.

In this study, we describe the genome sequence of a novel double-stranded RNA (dsRNA) mycovirus, designated as "Rhizoctonia solani partitivirus 15" (RsPV15), from the phytopathogenic fungus Rhizoctonia solani. RsPV15 consists of two genomic double-stranded RNA segments, dsRNA-1 and dsRNA-2, which are 2433 bp and 2350 bp long, respectively. Each of the dsRNA segments contains a single open reading frame, encoding the putative RNA-dependent RNA polymerase and coat protein, respectively. Homology searches and phylogenetic analysis suggested that RsPV15 is a new member of the genus Betapartitivirus within the family Partitiviridae.

[Study on interaction between ginsenosides Rg_1,Rb_1 and Ro and bovine serum albumin].

Small molecules with physiological or pharmacological activities need to interact with biological macromolecules in order to function in the body. As the protein with the highest proportion of plasma protein,serum albumin is the main protein binding to various endogenous or exogenous small molecules. Serum albumin interacts with small molecules in a reversible non-covalent manner and transports small molecules to target sites. Bovine serum albumin( BSA) is an ideal target protein for drug research because of its low cost and high homology with human serum albumin. The research on the interaction between drugs and BSA has become a hotspot in the fields of pharmacy,medicine,biology and chemistry. In this research,molecular docking method was used to study the interaction between three small ginsenosides with high pharmacological value( Rg_1,Rb_1,Ro) and bovine serum albumin( BSA),and the binding mode information of three ginsenosides interacting with BSA was obtained. The results of molecular docking showed that ginsenosides and amino acid residues in the active pocket of proteins could be combined by hydrophobic action,hydrogen bonding and electrostatic action. The interaction between small ginsenosides and bovine serum albumin is not the only form,and their interaction has many forms of force. The interaction between these molecules and various weak forces is the key factor for the stability of the complex. The results of this study can provide the structural information of computer simulation for the determination of the interaction patterns between active components and proteins of ginseng.

Highly Enantioselective Construction of Hajos-Wiechert Ketone Skeletons via an Organocatalytic Vinylogous Michael/Stetter Relay Sequence.

A highly enantioselective supramolecular iminium-catalyzed vinylogous Michael addition/Stetter relay sequence has been developed. This transformation provided a series of Hajos-Wiechert-type fused bicyclic diones with three continuous stereogenic centers in good yields with excellent enantioselectivities. The obtained products can be easily transformed into other structures with potential synthetic value.

Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes.

STUDY DESIGN: Intermittent Cyclic Mechanical Tension (ICMT) was applied to end plate chondrocytes by using an FX-4000T Flexercell Tension Plus unit (Flexcell International Corporation, Hillsborough, NC). Changes of end plate chondrocytes were observed after ICMT stimulation. OBJECTIVE: To investigate the relationship between mechanical stimulation and calcification of end plate chondrocytes. SUMMARY OF BACKGROUND DATA: Previous study showed that end plate calcification was related to mechanical stress, but there was no clear evidence to indicate whether or not mechanical stimulation could induce calcification of end plate chondrocytes in vitro. METHODS: Rat end plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) was applied for 25 days, 4 hours a day and continued to culture for 5 days. End plate chondrocytes were incubated for 12 hours in the presence or absence of 10 ng/mL of transforming growth factor-ß1 (TGF-ß1) (prepared from a stock solution at 10 µg/mL in 2 mM citric acid containing 2 mg/mL bovine serum albumin) in MEM/F-12 containing a final concentration of 1% FCS. End plate chondrocytes calcification was stained by alizarin red S (AR-S). End plate chondrocytes viability was examined by LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA). Related gene expression was examined by reverse transcription-polymerase chain reaction and Western blot. RESULTS: LIVE/DEAD assay verified that the nonloading (NC) group and the ICMT group end plate chondrocytes remained adherent, with no change in viability after the application of ICMT. Alizarin red staining showed that ICMT induced the calcification of end plate chondrocytes. Real-time reverse transcription-polymerase chain reaction showed that mRNA expression of endogenous TGF-ß1 decreased and mRNA expression of type I, type X, osteocalcin and osteopontin increased after ICMT. The ankh gene expression of both mRNA and protein levels decreased in the ICMT stimulation. The ankh gene expression of both mRNA and protein levels increased in TGF-ß1 stimulation. Compared with NC group, the alkaline phosphatase activities significantly increased in ICMT group. CONCLUSION: Our results directly showed that ICMT induced the calcification and downregulation of ankh gene expression of end plate chondrocytes, which may be caused by the endogenous TGF-ß1.

[Significance and expression of Sirt1 gene in human cervical endplate chondrocyte in vitro degeneration].

OBJECTIVE: To observe the expression changes of Sirt1 gene and examine the role and significance of degenerative process in human cervical endplate chondrocytes through a degeneration model of human cervical vertebral endplate chondrocyte. METHODS: Cartilage endplates of 30 patients were divided into control group (n = 16) with cervical vertebral fracture or dislocation and cervical spondylosis group (n = 14) with cervical spondylotic myelopathy. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro for 10 days. The differences of endplate chondrocytes from normal and degenerative cartilage endplates were observed by inverted phase-contrast microscope, hematoxylin and eosin staining and toluidine blue staining. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA expressions of Sirt1, collagen II and aggrecan. RESULTS: Compared with the normal group, the cellular morphology of degenerative group showed spindle-shaped changes. The mRNA expression of Sirt1 (P = 0.034) significantly decreased. Aggrecan (P = 0.0063) and collagen II (P = 0.0072) decreased also markedly. CONCLUSION: Sirt1 gene expression is significantly down-regulated in degenerative human cervical endplate chondrocytes. Regulating the expression of Sirt1 gene may block or delay the occurrence of human cervical endplate cartilage degeneration.

[Altered expression of endogenous transforming growth factor ß1 and early calcification related genes in rat endplate].

OBJECTIVE: To explore the relationship between endogenous transforming growth factor (TGF)-ß1 and calcification-related genes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process. METHODS: Endplate chondrocytes were extracted from rat lumbar vertebrae, isolated by enzyme digestion and P2 and P4 generations selected for a 6-day in vitro culture. The specimens were photographed microscopically to observe the cellular differences by alizarin red staining. Type II collagen marker gene, transcription factor SOX-9 gene and metabolism-related genes proteoglycan. matrix metalloproteinase (MMP)-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 were detected by RT-PCR to verify the degeneration model. Based on this model, the changes of growth factor TGF-ß1 and calcification-related genes ankyrin (ANK), ectonucleotide pyrophosphatase (ENPP), tissue-nonspecific alkaline phosphatase (TNAP) were continuously tested. RESULTS: Compared with P2 cells, P4 cells tended to assume a spindle-shaped morphology. And alizarin red staining showed no change between them. The level of transcription factor SOX-9 of P4 cells (P4/P2 = 0.0690, P = 0.0489) was significantly lower than that of P2 cells. Type II collagen (P4/P2 = 0.0535, P = 0.009) and proteoglycan (P4/P2 = 0.2672, P = 0.0343) were also significantly lower than those of P2 cells. No significant changes were observed in other metabolism-related genes. TGF-ß1 (P4/P2 = 0.5934, P = 0.0482) was significantly lower. The expressions of TNAP (P4/P2 = 0.0385, P = 0.0139) and ANK (P4/P2 = 0.2121, P = 0.0009) were significantly lower. But ENPP showed no significant change. CONCLUSION: P4 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number. Type II collagen, SOX-9 and proteoglycan are significantly reduced. Endogenous TGF-ß1 gene and calcification-related genes are down-regulated. The decrease of ANK gene may be caused by the down-regulation of endogenous TGF-ß1. Modulating the expression of endogenous TGF-ß1 gene in endplate chondrocytes may become a new therapeutic approach for the degeneration of intervertebral disc.