Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases

The replication enzyme of RNA viruses must preferentially recognize their RNAs in an environment that contains an abundance of cellular RNAs. The factors responsible for specific RNA recognition are not well understood, in part because viral RNA synthesis takes place within enzyme complexes associated with modified cellular membrane compartments. Recombinant RNA-dependent RNA polymerases (RdRps) from the human norovirus and the murine norovirus (MNV) were found to preferentially recognize RNA segments that contain the promoter and a short template sequence for subgenomic RNA synthesis. Both the promoter and template sequence contribute to stable RdRp binding, accurate initiation of the subgenomic RNAs and efficient RNA synthesis. Using a method that combines RNA crosslinking and mass spectrometry, residues near the template channel of the MNV RdRp were found to contact the hairpin RNA motif. Mutations in the hairpin contact site in the MNV RdRp reduced MNV replication and virus production in cells. This work demonstrates that the specific recognition of the norovirus subgenomic promoter is through binding by the viral RdRp.     

Muscarinic receptors in the cerebellar vermis modulate the gain of the vestibulospinal reflexes in decerebrate cats.

1. The Purkinje (P)-cells of the cerebellar vermis, which exert a prominent influence on posture as well as on the gain of vestibulospinal (VS) reflexes, are under the control not only of the classic mossy fibers and climbing fibers which liberate excitatory amino acids as neurotransmitter, but also of cholinergic afferents. The role of these afferents was investigated in precollicular decerebrate cats by using the method of local microinjection of cholinergic agents into appropriate areas of the cerebellar cortex. 2. Unilateral injection into the vermal cortex of the culmen of the non-selective cholinergic agonist carbachol (0.25 microliters at 0.5 micrograms/microliters saline) produced a postural asymmetry, characterized by a slight decrease of the extensor tonus in the ipsilateral forelimb and an increased tonus in the contralateral forelimb. Moreover, the gain of the EMG responses of the ipsilateral and the contralateral triceps brachii to animal tilt increased significantly, while no significant changes in the phase angle of the responses were observed. These effects started 5-10 min after the injection and persisted for at least 2 hours before disappearing. Similar but smaller effects were obtained after injection of eserine, an inhibitor of acetylcholinesterase. Thus, the effects could be produced by increasing the naturally present amount of acetylcholine (ACh). 3. The changes in posture and gain of the VS reflexes described above utilized in part at least muscarinic receptors, since effects similar to those induced by carbachol injection were also obtained after unilateral microinjection into the vermal cortex of the culmen of the muscarinic agonist bethanechol (0.25 microliters at 0.1 micrograms/microliters). On the other hand opposite effects, characterized by an increased postural activity in the ipsilateral forelimb associated with a decreased activity in the contralateral forelimb, as well as by a reduced gain of the EMG responses of the triceps brachii of both sides to animal tilt were observed in other experiments after local microinjection of the muscarinic antagonist scopolamine (0.25 microliter at 4-8 micrograms/microliters saline). Evidence for muscarinic supersensitivity was obtained following repetitive injections of scopolamine into the cerebellar vermis. 4. The area which upon injection of the cholinergic agents modified the postural activity as well as the gain of the VS reflexes was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.4-1.8 mm with respect to the midline.(ABSTRACT TRUNCATED AT 400 WORDS)     

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7xrace 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Avr4 and Avr6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F(2) population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0cM distant from the Avr4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Avr4/6 locus. The chromosome walk spanned a physical distance of 67kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Avr4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Avr4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Avr4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24kb and 4.3cM that appears to include the Avr4/6 locus.     

Craniorachischisis in a squirrel monkey

A stillborn squirrel monkey had craniorachischisis. The calvarium was absent and the skin and vertebral neural arches were cleft from the foramen magnum to the midlumbar region. Cranial nerves and degenerating neural tissue were attached to the cranial base and there were remnants of spinal cord in skin-covered areas of the spinal canal. The adrenal glands were normal. This is the only reported case of craniorachischisis in a monkey fetus.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.