Enzymatically inactive adenylate kinase 4 interacts with mitochondrial ADP/ATP translocase

Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H₂O₂ induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H₂O₂ treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.     

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.RNA viruses are able to undergo rapid genetic changes in order to adapt to new hosts or environments. Although much of this flexibility is due to the error-prone nature of the RNA-dependent RNA polymerase, which generates an array of different point mutations within the viral population (23), recombination is also a common and important mechanism for generating viral diversity (18, 31, 42, 58). Recombination occurs when the RNA-dependent RNA polymerase switches templates during replication, an event that is favored when both templates share identical or very similar sequences. Three types of RNA recombination have been identified: homologous recombination occurs at sites with exact sequence matches; aberrant homologous recombination requires sequence homology, but crossover occurs either upstream or downstream of the site of homology, resulting in a duplication or deletion; and nonhomologous (or illegitimate) recombination is independent of sequence homology (31, 42).When the same cell is infected by viruses of two different strains, or even different species, recombination between their genomic RNAs can potentially lead to the emergence of new pathogens. A case in point is the emergence of Western equine encephalitis virus, a member of the genus Alphavirus, family Togaviridae, which arose by homologous recombination between Eastern equine encephalitis virus and Sindbis virus (14).Some mammalian RNA viruses can recombine at a frequency that is detectable in experimental settings (1, 2, 55), and phylogenetic analysis of partial or complete genome sequences suggests that RNA recombination is a widespread phenomenon. Naturally occurring recombinant viruses have been identified in almost every family of positive-stranded RNA viruses (31, 58).Flaviviruses are members of the genus Flavivirus, family Flaviviridae, a family that also includes the genera Pestivirus and Hepacivirus. Several of the flaviviruses are important human pathogens, such as Japanese encephalitis virus (JEV), West Nile virus (WNV), the dengue viruses, yellow fever virus, and tick-borne encephalitis virus (TBEV).Although there has never been a report of a pathogenic flavivirus strain arising due to recombination involving attenuated vaccine strains (39), the urgent necessity to develop tetravalent vaccines containing all four serotypes of dengue virus—two such vaccines are currently undergoing clinical testing (45)—has recently brought the recombination issue to the forefront of discussion among researchers, regulators, and vaccine producers (39). It has been suggested that recombination, either between the strains present in a multivalent vaccine or between an attenuated vaccine strain and a wild-type strain, could lead to the emergence of new viruses with unpredictable properties (49).So far, recombination between flavivirus genomes has not been demonstrated directly in the laboratory. However, phylogenetic analysis of partial genome sequences available in the GenBank database has suggested that homologous recombination may have occurred between closely related strains of dengue virus (20, 52, 54, 59). An experimental approach for assessing the ability of flavivirus genomes to recombine is therefore urgently needed.Flavivirus virions are composed of a single-stranded, positive-sense RNA genome that, together with the capsid protein C, forms the viral nucleocapsid. The nucleocapsid is covered by a lipid envelope containing the surface glycoproteins prM and E. These glycoproteins drive budding at the membrane of the endoplasmic reticulum during the assembly stage and mediate entry of the virus into host cells (41). Replicons, defined as self-replicating, noninfectious RNA molecules, can be generated by deleting parts or all of the region coding for the structural proteins C, prM, and E from the viral genome but maintaining all seven of the nonstructural proteins and the flanking noncoding sequences, which are required in cis for RNA replication (25). By providing the missing structural protein components in trans, replicons can be packaged into virus-like particles that are capable of a single round of infection (10, 15, 24, 47).Typically, researchers developing novel replicating vaccines, especially ones that involve multiple components, make an effort to come up with strategies to prevent recombination, for example by “wobbling” codons, i.e., replacing codons in homologous regions with synonymous ones encoding the same amino acid but consisting of a different nucleotide triplet (50, 57). In this study, in order to assess the propensity of flavivirus genomes to recombine, we took an opposite approach, establishing a “recombination trap” that favors the selection and sensitive detection of recombination products. This system takes advantage of the ability of replicon pairs containing deletions in their structural protein genes to complement each other in trans and thus be propagated together in cell culture, and by passage at limiting dilutions, it allows infectious RNA genomes arising by recombination between the two replicons to be preferentially selected.Using the recombination trap, we have now obtained the first direct evidence of recombination between flavivirus genomes in the laboratory. Aberrant homologous recombination was observed twice with JEV replicons, resulting in viruses with unnatural gene arrangements and reduced growth properties compared to those of wild-type JEV. No infectious recombinants of any kind were obtained when TBEV or WNV replicons were used. Interestingly, we never detected a fully infectious wild-type genome arising by homologous recombination in any of these systems. The results of this study show that the propensity of flavivirus genomes to recombine in the region coding for the structural proteins appears to be quite low, suggesting that recombination does not represent a major risk in the use of live, attenuated flavivirus vaccines.     

Motor network degeneration in amyotrophic lateral sclerosis: a structural and functional connectivity study

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by motor neuron degeneration. How this disease affects the central motor network is largely unknown. Here, we combined for the first time structural and functional imaging measures on the motor network in patients with ALS and healthy controls.

Methodology/Principal Findings

Structural measures included whole brain cortical thickness and diffusion tensor imaging (DTI) of crucial motor tracts. These structural measures were combined with functional connectivity analysis of the motor network based on resting state fMRI. Focal cortical thinning was observed in the primary motor area in patients with ALS compared to controls and was found to correlate with disease progression. DTI revealed reduced FA values in the corpus callosum and in the rostral part of the corticospinal tract. Overall functional organisation of the motor network was unchanged in patients with ALS compared to healthy controls, however the level of functional connectedness was significantly correlated with disease progression rate. Patients with increased connectedness appear to have a more progressive disease course.

Conclusions/Significance

We demonstrate structural motor network deterioration in ALS with preserved functional connectivity measures. The positive correlation between functional connectedness of the motor network and disease progression rate could suggest spread of disease along functional connections of the motor network.     

Classification of large array surface myoelectric potentials from subjects with and without low back pain.

An algorithm was developed and tested for differentiating between the spatial distribution of large arrays of surface electromyographic (LASE) data from subjects with and without low back pain (LBP). The surface EMG data from 62 channels were collected from the low back of 161 healthy and 44 acute (less than 6-weeks) LBP subjects in three minimum stress postural positions including standing, 20 degrees of trunk flexion (at hip joint) and standing with arms extended forward holding a 1.36kg (3lb) weight in each hand. These data were statistically analyzed and the spatial distribution of the root mean square (RMS) values was used in a multivariate quadratic discriminant model to reclassify the healthy and acute LBP subjects. The most predictive results were obtained from the 'flexion' group of experiments and correctly reclassified 95.5% (42/44) of the acute LBP subjects and 99.4% (160/161) of the healthy subjects. The success rate of this reclassification based on surface distribution of myoelectric potentials was found to be better than the reported patient classifications based on a smaller set of electrode pairs using fewer subjects [Peach JP, McGill SM, Classification of low back pain with use of spectral electromyogram parameters. Spine 23(10):1998;1117-23; Roy SH, De Luca CJ, Emley M, Oddsson LI, Buijs RJ, Levins JA, Newcombe DS, Jabre JF. Classification of back muscle impairment based on the surface electromyographic signal. J Rehabil Res Dev 34(4):1997;405-14 [review]]. The results indicated the potential of the model for clinical patient classification.     

Preferential transport of glutathione versus glutathione disulfide in rat liver microsomal vesicles

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer.     

VEGF is a chemoattractant for FGF-2-stimulated neural progenitors

Migration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.     

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.     

Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations

The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1. Orcinol was used as an inducer for enzyme production. The growth medium was optimized in two steps. In the first step, screening for optimal glucose concentration was performed. In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium. After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium. Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods. In a subsequent step, the maximal orcinol-degradation rate was determined. The results obtained were used to find an optimal feeding profile for enzyme production. By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments. Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).