Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.     

A novel approach to intrauterine viral inoculation of swine using PCV type 2 as a model

Experimentally intrauterine (IU) viral inoculation has been commonly used to circumvent maternal interference with transplacental infection of fetuses and to assess the effect of viral infection on fetal development or reproductive parameters. However, IU inoculation requires surgical procedures such as laparatomy and surgical incision of the uterus. Post-surgical complications, that frequently result in abortion or fetal death, have been a major disadvantage. An animal trial was conducted to evaluate the non-surgical procedure of ultrasound needle-guided transabdominal injection for IU inoculation of porcine circovirus type 2 (PCV2) since this virus has been reported to cause reproductive failure in pigs. Two groups of seven pregnant sows at mid- and late-gestation, respectively, were inoculated with PCV2 using an ultrasound needle-guided technique that delivered PCV2 directly into one of the fluid-filled fetal compartments. The effect of transabdominal in utero virus challenge on fetuses and sows was assessed until term. While five of six sham-inoculated control sows had no or minimal adverse affects from in utero injection, 10 of 14 virus-inoculated sows had dead and/or stillborn piglets and PCV2 infection was evident by polymerase chain reaction and/or immunohistochemistry. These results supported previous field and experimental observations that PCV2 may cause reproductive failure. In conclusion, ultrasound needle-guided transabdominal injection was a safe and efficient method for IU inoculation of virus in pigs.     

Solid state and solution studies of a vanadium(III)-L-cysteine compound and demonstration of its antimetastatic, antioxidant and inhibition of neutral endopeptidase activities

Reaction of one equivalent of vanadium(III) chloride with three equivalents of l-cysteine(H2Cys) in methyl alcohol affords a VIII-Cys compound that is formulated as [VIII(Hcys)3].2HCl.2.5H2O 1. The solid state characterization of 1 was performed by microanalysis, circular dichroism (CD) and infrared studies as well as room temperature magnetic susceptibility. These studies have shown coordination of each HCys- ligand to the VIII atom through an amine nitrogen and a carboxylate oxygen atoms. Solution studies of 1 were carried out in water and methanol by UV-visible, CD and electron paramagnetic resonance (EPR) spectroscopies. According to these studies, it was evident that despite the progressive oxidation of 1 to oxovanadium(IV) species, some V(III) species were also present in solution after several hours. Compound 1, VIVOSO4.5H2O and l-cysteine were examined for their total antioxidant capacity (TAC) and lag time. Compound 1 exhibited significantly greater total antioxidant capacity and lag time values than l-cysteine. VIVOSO4.5H2O did not show any total antioxidant capacity or lag time. The inhibition of neutral endopeptidase (NEP) activity caused by 1, VIVOSO4.5H2O and thiorphan was also measured. Compound 1, at a concentration of 10(-3) M, showed inhibition of NEP activity as potent as thiorphan at 10(-6) M, while VIVOSO4.5H2O in the same concentration exhibited less than 50% inhibitory activity than that of thiorphan at 10(-6) M. Moreover, the antimetastatic effects of compound 1, l-cysteine and VIVOSO4.5H2O were examined on Wistar rats, treated with 3,4-benzopyrene. The results revealed that 1 prevents significantly lung metastases (only 9.5% of animals treated with 1 showed metastases), whereas 47-52% of the rats of the control group and those treated with l-cysteine and VIVOSO4.5H2O exhibited metastases.     

Our experiences with the treatment of periprosthetic fractures of femur

In the statement authors want to draw attention to the possibilities of treatment of periprosthetic fractures of femur. They present their own experiences with the treatment of these fractures by using various types of internal and external fixation evaluated from 1996 to 2003. They present some less common types of internal fixation as e.g. fixation by clamp plates.     

Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo

A moderate genotoxic activity of halothane and isoflurane applied as volatile anaesthetics has already been shown. The aim of this work was to estimate a potential genotoxicity of sevoflurane, introduced to clinical practice later than halothane and isoflurane. A genotoxic activity of all three compounds was estimated by using the comet assay in human peripheral blood lymphocytes (PBL) proliferating in vitro. We demonstrated that in contrast to the previously studied anaesthetics, sevoflurane did not induce any increase in DNA migration in the studied conditions. To estimate a genotoxic effect of a prolonged exposure to halogenated anaesthetics in vivo, PBL taken from operating room personnel (n = 29) were tested for DNA degradation and compared with those from a control non-exposed group (n = 20). No significant differences were detected between the groups. We conclude that sevoflurane does not have genotoxic properties, both in vitro and in vivo.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway

Epithelial cell polarity depends on mechanisms for targeting proteins to different plasma membrane domains. Here, we dissect the pathway for apical delivery of several raft-associated, glycosyl phosphatidylinositol (GPI)-anchored proteins in polarized MDCK cells using live-cell imaging and selective inhibition of apical or basolateral exocytosis. Rather than trafficking directly from the trans-Golgi network (TGN) to the apical plasma membrane as previously thought, the GPI-anchored proteins followed an indirect, transcytotic route. They first exited the TGN in membrane-bound carriers that also contained basolateral cargo, although the two cargoes were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that transcytosed to the apical plasma membrane. Thus, apical sorting of these GPI-anchored proteins occurs at the plasma membrane, rather than at the TGN.     

The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.     

Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.