OBJECTIVE

The cyclin-dependent kinase inhibitor, seliciclib (R-roscovitine, CYC202), has anti-proliferative activity through its inhibition of cyclin-dependent kinase 2. We hypothesized that treatment with seliciclib would reduce glomerular macrophage numbers and glomerular crescent formation in experimental crescentic glomerulonephritis even when treatment is started after onset of disease.

METHODS

Nephrotoxic nephritis (NTN) was induced in Wistar Kyoto rats. In experiment 1, seliciclib (150 mg/kg per day) was given by oral gavage from 1 h before induction of NTN and continued to day 14. In experiment 2, treatment was started on day 4 of NTN and continued to day 14 in order to examine the effect of seliciclib in established glomerulonephritis.

RESULTS

In experiment 1, seliciclib reduced proteinuria (119.5 ± 13.9 vs 191.4 ± 18.8 mg/day, P < 0.01), serum creatinine (54.0 ± 3.0 vs 81.0 ± 2.5 µmol/L, P < 0.005) and glomerular crescent score (23.9 ± 2.1 vs 44.6 ± 2.2, P < 0.005) in comparison with controls. In experiment 2, seliciclib ameliorated established glomerulonephritis, with reduction in proteinuria (58 ± 16 vs 165 ± 13 mg/day, P < 0.005), serum creatinine (39 ± 3 vs 62 ± 5 µmol/L, P < 0.05), glomerular macrophage numbers (6.8 ± 2.5 vs 18.5 ± 1.2 ED1+ cells per glomerular cross section, P < 0.05), glomerular cell proliferation (1.2 ± 0.37 vs 4.2 ± 0.80 bromodeoxyuridine (BrdU)+ cells per glomerular section, P < 0.05) and crescent score (10.8 ± 1.6 vs 43.9 ± 1.4, P < 0.05), in comparison with the controls.

CONCLUSIONS

Seliciclib is effective in both prevention and treatment of established crescentic glomerulonephritis in Wistar Kyoto rats, in association with a reduction in the number of glomerular macrophages. We suggest that seliciclib, or other cyclin-dependent kinase inhibitors, may represent a novel therapeutic approach for patients with proliferative glomerulonephritis.

This study evaluated the effects of supplementation of carnitine and antioxidants on lipids, carnitine concentrations, and exercise endurance time in both trained and untrained rats as compared to non-supplemented rats. Thirty-two male SD rats, age 7 wk were divided into four groups according to exercise training and modified AIN-76 diets: NTNS (non-trained non-supplemented), NTS (non-trained supplemented), LTNS (long-trained non-supplemented) and LTS (long-trained supplemented). The trained rats were run on a treadmill for 60 min per day (10(0) incline, 20 m/min for 8 wk). Carnitine (0.5%/diet) and vitamin E (0.5 mg/g b.w.) were supplemented in rat diets and vitamin C (0.5 mg/g b.w.) and melatonin (1 microg/g b.w.) were administered into the stomachs of the rats. LTNS and LTS rats had significantly lower serum total lipid, triglyceride, total cholesterol and liver triglycerides, but had higher serum HDL-cholesterol. There were no changes in exercise endurance time by supplementation in untrained animals, however endurance times were longer in LTS animals than in LTNS. The supplementation and training tended to increase carnitine palmitoyltransferase (CPT-I) activities, although the differences were not statistically significant. Likewise, CPT-I mRNA levels were higher in both supplemented and exercise trained rats. These results suggest that supplementation of carnitine and antioxidants may improve lipid profiles and exercise ability in exercise-trained rats.

GLIAL cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) are members of a new family of factors within the transforming growth factor-beta (TGFbeta) super-family. Signaling by GDNF and NTN depends on the tyrosine kinase receptor Ret and on GFRalpha-1 and -2. We have investigated by competitive RT-PCR the developmental expression of these molecules within the rat hippocampus. All transcripts reach high expression levels between postnatal day 0 and 14 (P0-P14), a critical period of hippocampal differentiation, suggesting roles for these molecules in this process. All mRNAs (with the exception of Ret) can be detected as early as embryonic day 14 (E14). The absence of Ret expression at E14 suggests that both GDNF and NTN may employ an additional, unknown receptor. NTN expression shows a biphasic expression pattern, with maximum expression levels at E14 and P5.

The glial cell line-derived neurotrophic factor (GDNF) family of ligands binds to lipid anchored proteins termed GDNF family receptor (GFR)alphas, and then activates the RET receptor tyrosine kinase, by ligand GFRalpha. The binding of soluble GFRalphas to transfected cells suggested that different GFRalphas were dedicated to particular ligands, with GDNF acting primarily or entirely through GFRalpha1, and neurturin (NTN), through GFRalpha2. More recent evidence has suggested the possibility of cross-talk between these ligands and the two receptors. We examined here whether crosstalk between the GDNF ligands and the GFRalphas is biologically relevant, using midbrain dopaminergic, and parasympathetic, submandibular gland neurons. By biochemical and genetic addition and/or deletion of GFRalpha1 and 2, we show that in both neuronal cell types, robust biological activities of GDNF or NTN can be mediated by either GFRalpha1 or GFRalpha2, although GDNF is slightly more potent in dopaminergic (DA) neurons which normally express GFRalpha1, and NTN in submandibular neurons which normally express GFRalpha2. Throughout the body, GDNF and NTN are likely to have important biological actions on both GFRalpha1- and GFRalpha2-expressing cells.

Extreme resistance in cultivated potato (Solanum tuberosum) to potato viruses Y and A (PVY and PVA) conditioned by the presence of Ry genes introduced from Solanum stoloniferum was described by Cockerham (1970). Cockerham detailed a number of genes which controlled a variety of reactions, including extreme resistance to both viruses (i.e. little or no visible reaction of plants and no viral replication following graft and manual inoculation) controlled by gene Ry sto. In the present study, cvs 'Pirola' and 'Barbara', which contain a Ry gene, were found to have extreme resistance to PVY isolates from the ordinary (PVY°), veinal necrosis (PVY(N)) and potato tuber necrotic ringspot (PVY(NTN)) subgroups, and PVA. The inheritance of this phenotype was examined in seedling progenies obtained by crossing 'Barbara' and 'Pirola' with susceptible cultivars. Segregation data for resistance to PVY and PVA in a progeny involving cv 'Pirola' best fitted a genetical model of one gene controlling extreme resistance to both PVY and PVA, although the possibility that there are two genes, each controlling resistance to one virus but closely linked, cannot be excluded. Segregation data from progenies involving cv 'Barbara' best fitted a genetical model in which there are two independent genes, one controlling extreme resistance to PVA and PVY and a second gene controlling extreme resistance to PVA but not to PVY. This previously unrecognised gene conferring extreme resistance to PVA only, should be given the notation Ra in keeping with nomenclature used for other resistance genes.

The reactive oxygen species hydrogen peroxide (H(2)O(2)) was detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) 24 and 48 h after a Potato virus Y (PVY) infection. Hydrogen peroxide was detected in vivo by its reaction with 3.3-diaminobenzidine, producing a reddish-brown staining in contact with H(2)O(2). Hydrogen peroxide was detected in the necrotic area of the epidermal and mesophyll cells 24 and 48 h after the PVY infection. Highly localised accumulations of H(2)O(2) were found within xylem tracheary elements, and this was much more intensive than in non-infected leaves. Hydrogen peroxide was detected cytochemically in HR also by its reaction with cerium chloride, producing electron-dense deposits of cerium perhydroxides. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised accumulations of H(2)O(2) was detected in plant cell walls. The most intensive accumulation was present in the bordering cell walls of necrotic mesophyll cells and the adjacent non-necrotic mesophyll cells. Intensive electron-dense deposits of cerium perhydroxide were found along ER cistrenae and chloroplast envelopes connected with PVY particles. The precipitates of hydrogen peroxide were detected in the nuclear envelope and along tracheary elements, especially when virus particles were present inside. The intensive accumulation of H(2)O(2) at the early stages of potato-PVY interaction is consistent with its role as an antimicrobial agent and for this reason it has been regarded as a signalling molecule.

Glial cell line-derived neurotrophic factor family receptor alpha-2 (GFR alpha-2) is a GPI-linked receptor that preferentially binds neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family. Three splice isoforms of GFR alpha-2 have been identified previously in mouse tissues, but the occurrence of splice isoforms in rats has not been described. The aim of this study was therefore to identify GFR alpha-2 splice isoforms in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and gene cloning. Three isoforms were identified and sequenced, and named GFR alpha-2(a), (b) and (c), according to the nomenclature used for the previously identified mouse isoforms. The GFR alpha-2(a) and (b) isoforms were identical to those previously described in mice. The GFR alpha-2(c) isoform was novel. Sequences for GFR alpha-2(b) and (c) were deposited in the GenBank database (accession numbers GI: 16797788 and 16797786, respectively). All three isoforms were expressed in the brain, kidney, and intestine of both postnatal and adult rats.

The effects of vitamin E on tissue oxidation, kidney function and morphology were studied in rats with nephrotoxic nephritis (NTN). Thirty-six nephritic animals received no treatment (group 1), while 36 were treated with vitamin E (group 2). Twenty-four hours after the administration of anti-glomerular basement membrane antibody, sulfhydryl-containing renal protein was significantly lower in group 1 than in group 2 (0.70 +/- 0.16 and 1.08 +/- 0.06 mmol/100 g kidney tissue, respectively), suggesting a free oxygen radical scavenging effect of vitamin E in group 2. The difference was similar on day 14. The creatinine clearance was significantly lower in group 1 than in group 2 on day 1 (40 +/- 30 and 204 +/- 60 microliter/min per 100 g body weight, respectively). The protein excretion was initially high in both groups, but a significant decrease was detected in group 2 relative to group 1 on day 14 (25 +/- 18 and 92 +/- 38 mg/24 h, respectively). The morphological changes were less severe in group 2. Vitamin E treatment did not alter any of the above values significantly in healthy animals. The release of oxygen free radicals in NTN might play an important role in the pathogenesis, which can be influenced by free radical scavengers through changes in kidney function and morphology.

Neurturin (NTN) is a recently characterized member of the glial cell line-derived neurotrophic factor (GDNF)-family which, like GDNF, can promote the survival of certain populations of neuronal cells in peripheral and central nervous systems. To elucidate the roles of NTN and a novel glycosyl-phosphatidylinositol (GPI)-linked receptor protein GFRalpha-3, a member of GDNF-family receptor alpha, in the regulation of peripheral trigeminal innervation and tooth formation, their expression patterns during mouse embryonic (E) and early postnatal (P) development (E10-P5) of the first branchial arch were analyzed by in situ hybridization. NTN mRNAs were observed in oral and cutaneous epithelia of the mandibular process at all studied stages and expression became gradually restricted to the suprabasal epithelial cells. In addition, transcripts were also detected in the epithelium of whisker follicles. In the developing first molar tooth germ, NTN showed a developmentally regulated, spatiotemporally changing expression pattern, which partially correlated with the development of innervation. During the initiation of tooth formation NTN mRNAs were expressed in dental epithelium and during later embryonic development transcripts appeared in the dental papilla mesenchyme. In addition, some transcripts were seen in the dental follicle. During postnatal development, NTN expression was restricted to the dental follicle of the incisor tooth germs. GFRalpha-3 mRNAs were not detected in teeth, but an intense expression was seen in non-neuronal cells surrounding trigeminal nerve fibers and in the trigeminal ganglia during E11-E15. Ganglion explant cultures showed that trigeminal neurons start to respond to exogenous NTN at E12, which correlates to the earlier reported appearance of the Ret-tyrosine kinase receptor in the trigeminal ganglion. Local application of NTN with beads on isolated dental mesenchyme did not stimulate cell proliferation or prevent apoptotic cell death. In addition, exogenous NTN had no effects on tooth morphogenesis in in vitro cultures. Taken together, because trigeminal neurons respond to NTN after first axons have reached their primary epithelial target fields, NTN is apparently not involved in the guidance of pioneer trigeminal nerves to their peripheral targets. However, our results show that NTN is a potent neuritogenic factor and, therefore, may act as a target-field-derived neurotrophic factor for trigeminal nerves during innervation of the cutaneous and oral epithelia as well as dental follicle surrounding the developing tooth. In addition, although NTN appears not to be directly involved in the regulation of tooth morphogenesis, it may have non-neuronal, organogenetic functions during tooth formation.

Penicillin V acylase (PVA), a member of newly evolved Ntn-hydrolase superfamily, is a pharmaceutically important enzyme to produce 6-aminopenicillanic acid. Active site characterization of recently purified monomeric PVA from Rhodotorula aurantiaca (Ra-PVA), the yeast source, showed the involvement of serine and tryptophan in the enzyme activity. Modification of the protein with serine and tryptophan specific reagents such as PMSF and NBS showed partial loss of PVA activity and substrate protection. Ra-PVA was found to be a multi-tryptophan protein exhibiting one tryptophan, in native and, four in its denatured condition. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. The results obtained indicate that the tryptophan residues of Ra-PVA were largely buried in hydrophobic core of the protein matrix. Quenching of the fluorescence by acrylamide was collisional. Acrylamide was the most effective quencher amongst all the used quenchers, which quenched 71.6% of the total intrinsic fluorescence of the protein, at a very less final concentration of 0.1M. Surface tryptophan residues were found to have predominantly more electropositively charged amino acids around them, however differentially accessible for ionic quenchers. Denaturation led to shift in lambda(max) from 336, in native state, to 357 nm and more exposed to the solvent, consequently increase in fluorescence quenching with all quenchers. This is an attempt towards the conformational studies of Ra-PVA.

Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: pepino mosaic virus (PepMV), potato virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.

Selective laser melting (SLM) has promising prospects in manufacturing customized implants, however the rough surface of SLM titanium specimen can facilitate bacterial adherence and biofilm formation, which is a risk to implant success. Therefore, surface modification is required to enhance its antibacterial efficacy. Sandblasting, anodization and electrochemical deposition were applied to construct a novel composite nanostructure of nanophase calcium phosphate embedded to TiO2 nanotubes on microrough SLM titanium substrates (NTN). NTN samples were compared with TiO2 nanotubes (NT) samples, mechanical polished (MP) samples and untreated SLM titanium samples. Surface characterization were analyzed using scanning electron microscope, energy dispersive spectroscopy, x-ray photoelectron spectroscopy, x-ray diffraction, a three dimensional profilometer and a contact angle measuring device. Bacteria adhesion assay for bacteria colony counting and bacteria LIVE/DEAD staining was conducted using Streptococcus mutans and Streptococcus sanguinis. Both S. mutans and S. sanguinis adherence on SLM samples were significantly higher than on NTN, NT and MP samples. The antibacterial efficacy of NTN samples was superior compared to NT and had no significant difference with MP samples, despite the fact that NTN samples had much rougher surface than MP samples. This study elucidates an efficient method to enhance antibacterial efficacy on rough SLM surfaces, which contributes to its application in dental and other biomedical implants.

Potato virus Y (PVY) is vectored by several potato-colonizing and non-colonizing aphid species in a non-persistent manner and has a wide host range. It occurs naturally in several plant families. Myzus persicae and Macrosiphum euphorbiae are the most efficient potato-colonizing aphid vectors of PVY. Rhopalosiphum padi, a cereal aphid that migrates in large numbers through potato fields during the middle of the growing season, does not colonize potato plants but can transmit PVY. Hairy nightshade, Solanum sarrachoides, a prevalent annual solanaceous weed in the Pacific Northwest (PNW) of the United States, is an alternative host for PVY and a preferred host for M. persicae and M. euphorbiae. Hence, hairy nightshade plants might play an important role as an inoculum source in the epidemiology of PVY. We looked at titre accumulation and distribution of PVY(O), PVY(N:O) and PVY(NTN) in S. sarrachoides and potato after aphid inoculation with M. persicae and studied the transmission of PVY(O) and PVY(NTN), by M. persicae, M. euphorbiae and R. padi from hairy nightshade to potato plants. Virus titre at different positions on the plant was similar in S. sarrachoides and potato plants with strains PVY(O) and PVY(N:O). Titres of PVY(NTN) were similar in S. sarrachoides and potato but differences in titre were observed at different positions within the plant depending on the plant phenology. Percentage transmission of PVY(NTN) by M. persicae and M. euphorbiae was twice as high (46 and 34%, respectively) from hairy nightshade to potato than from potato to potato (20 and 14%). Percentage transmission of PVY(O) by M. persicae and M. euphorbiae was not affected by the inoculum source. No effect of the inoculum source was observed in the transmission of either PVY strain by R. padi. These results show that hairy nightshade may be an equal or better virus reservoir than potato and thus, important in the epidemiology of PVY.

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease.

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVY^NTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVY^O (both PVY^O and PVY^O-O5), PVY^N, PVY^NA-N, PVY^NTN, PVY^Z, PVY^E, PVY-NE11, PVY^N-Wi, and PVY^N:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Computational fluid dynamic simulations of the flow in the Kyoto-NTN (Kyoto University, Kyoto, Japan) magnetically suspended centrifugal blood pump with a 16-straight-bladed impeller were performed in the present study. The flow in the pump was assumed as unsteady and turbulent, and blood was treated as a Newtonian fluid. At the impeller rotating speed of 2000 rpm and flow rate of 5 L/min, the pump produces a pressure head of 113.5 mm Hg according to the simulation. It was found that the double volute of the pump has caused symmetrical pressure distribution in the volute passages and subsequently caused symmetrical flow patterns in the blade channels. Due to the tangentially increasing pressure in the volute passages, the flow through the blade channels initially increases at the low-pressure region and then decreases due to the increased pressure. The reverse flow and vortices have been identified in the impeller blade channels. The high shear stress of the flow in the pump mainly occurred at the inlet and outlet of the blade channels, the beginning of the volute passages and the regions around the tips of the cutwater and splitter plate. Higher shear stress is obtained when the tips of the cutwater and splitter plate are located at the impeller blade trailing edges than when they are located at the middle of the impeller blade channel. It was found that the blood damage index assessed based on the blood corpuscle path tracing of the present pump was about 0.94%, which has the same order of magnitude as those of the clinical centrifugal pumps reported in the literature.

Overactivation of the renin-angiotensin system (RAS) - a central physiological pathway involved in controlling blood pressure (BP) - leads to hypertension. It is now well-recognized that the central nervous system (CNS) has its own local RAS, and the majority of its components are known to be expressed in the brain. In physiological and pathological states, the (pro)renin receptor (PRR), a novel component of the brain RAS, plays a key role in the formation of angiotensin II (Ang II) and also mediates Ang II-independent PRR signaling. A recent study reported that neuronal PRR activation is a novel mechanism for cardiovascular and metabolic regulation in obesity and diabetes. Expression of the PRR is increased in cardiovascular regulatory nuclei in hypertensive (HTN) animal models and plays an important role in BP regulation in the CNS. To determine the clinical significance of the brain PRR in human hypertension, we investigated whether the PRR is expressed and regulated in the paraventricular nucleus of the hypothalamus (PVN) and rostral ventrolateral medulla (RVLM) - two key cardiovascular regulatory nuclei - in postmortem brain samples of normotensive (NTN) and HTN humans. Here, we report that the PRR is expressed in neurons, but not astrocytes, of the human PVN and RVLM. Notably, PRR immunoreactivity was significantly increased in both the PVN and RVLM of HTN subjects. In addition, PVN-PRR immunoreactivity was positively correlated with systolic BP (sBP) and showed a tendency toward correlation with age but not body mass index (BMI). Collectively, our data provide clinical evidence that the PRR in the PVN and RVLM may be a key molecular player in the neural regulation of BP and cardiovascular and metabolic function in humans.

Keywords: (pro)renin receptor/(P)RR; human hypertension; paraventricular nucleus of the hypothalamus; renin-angiotensin system; rostral ventrolateral medulla.

Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome.

The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR.

The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato.

The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.

Immune-mediated glomerulonephritis is a serious end organ pathology that commonly affects patients with systemic lupus erythematosus (SLE). A classic murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN), in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important in the pathogenesis of LN. To further investigate the mechanism by which macrophages contribute to the pathogenic process, and to determine if this contribution is mediated by NF-κB signaling, we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression. Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly attenuated proteinuria, lower BUN levels, and improved renal histopathology compared to control injected wildtype B6 mice (WT). Inhibiting myeloid NF-κB signaling also decreased inflammatory modulators within the kidneys. We found significant decreases of IL-1a, IFNg, and IL-6 in kidneys from KO mice, but higher IL-10 expression. Flow cytometry revealed decreased numbers of kidney infiltrating classically activated macrophages in KO mice as well. Our results indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. While approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.

Snake venom phospholipases B (SVPLBs) are the least studied enzymes. They constitute about 1% of Bothrops crude venoms, however, in other snake venoms, it is present in less than 1%. These enzymes are considered the most potent hemolytic agent in the venom. Currently, no structural information is available about these enzymes from snake venom. To better understand its three-dimensional structure and mechanisms of envenomation, the current work describes the first model-based structure report of this enzyme from Bothrops moojeni venom named as B. moojeni phospholipase B (PLB_Bm). The structure model of PLB_Bm was generated using model building software like I-TESSER, MODELLER 9v19, and Swiss-Model. The build PLB_Bm model was validated using validation tools (PROCHECK, ERRAT, and Verif3D). The analysis of the PLB_Bm modeled structure indicates that it contains 491 amino acid residues that form a well-defined four-layer αββα sandwich core and has a typical fold of the N-terminal nucleophile aminohydrolase (Ntn-hydrolase). The overall structure of PLB_Bm contains 18 β-strands and 17 α-helices with many connecting loops. The structure divides into two chains (A and B) after maturation. The A chain is smaller and contains 207 amino acid residues, whereas the B chain is larger and contains 266 amino acid residues. The sequence and structural comparison among homologous snake venom, bacterial, and mammals PLBs indicate that differences in the length and sequence composition may confer variable substrate specificity to these enzymes. Moreover, the surface charge distribution, average volume, and depth of the active site cavity also vary in these enzymes. The present work will provide more information about the structure-function relationship and mechanism of action of these enzymes in snakebite envenomation.

Keywords: glycosylation; sequence and three-dimensional structure analysis; snake venom phospholipases B; structural comparison; structure-based substrate specificity and maturation.

Daily precipitation nitrate and ammonium concentration models were developed for the Chesapeake Bay Watershed (USA) using a linear least-squares regression approach and precipitation chemistry data from 29 National Atmospheric Deposition Program/National Trends Network (NADP/NTN) sites. Only weekly samples that comprised a single precipitation event were used in model development. The most significant variables in both ammonium and nitrate models included: precipitation volume, the number of days since the last event, a measure of seasonality, latitude, and the proportion of land within 8km covered by forest or devoted to industry and transportation. Additional variables included in the nitrate model were the proportion of land within 0.8km covered by water and/or forest. Local and regional ammonia and nitrogen oxide emissions were not as well correlated as land cover. Modeled concentrations compared very well with event chemistry data collected at six NADP/AirMoN sites within the Chesapeake Bay Watershed. Wet deposition estimates were also consistent with observed deposition at selected sites. Accurately describing the spatial distribution of precipitation volume throughout the watershed is important in providing critical estimates of wet-fall deposition of ammonium and nitrate.