The research group of Terumo Corporation, NTN Corporation, and the Setsunan University have been developing an implantable left ventricular assist system (T-ILVAS) featuring a centrifugal blood pump with a magnetically suspended impeller (MSCP). The present study describes results of chronic animal experiments using the MSCP. The MSCP has been tested ex vivo and in vivo in 6 sheep as a left heart bypass between the left ventricular apex and descending aorta. Ex vivo chronic sheep experiments using Model I demonstrated long-term durability, nonthrombogenicity, low hemolysis (<6 mg/dl), and excellent stability of the magnetic bearing with long-term survival for up to 864 days. Average pump flow rate was 4 L/min at a fixed rotational speed of 2000 rpm. Power spectral analyses of heart rate, aortic pressure, and blood temperature maintained normal 1/f fluctuation during the study. The retrieved pump was completely free from thrombus formation and there was no evidence of infarct in major organs. The implantable Model II was evaluated ex vivo in two sheep and intra-thoracically implanted in a sheep. These experiments were terminated at 70, 79, and 17 days due to blood leakage through the connector system within the housing. No thrombus formation was observed in any of the retrieved pumps. A modified Model II with a new connector system was subsequently intra-thoracically implanted in a sheep. The sheep survived for 482 days without any sign of thromboembolic complication or hemolysis at a fixed rotational speed of 1700 rpm and an average pump flow rate of 5 L/min. There was no intra-device thrombus formation or infarct in major organs. The Model III system, consisting of an implantable controller and a new MSCP with a reduced input power of 13 W, has been developed and implanted in a chronic sheep model. The MSCP was implanted in the left pleural space and the controller in the abdominal wall. The experiment is still in progress for more than 30 days without any significant complication to date. These animal studies strongly suggest the feasibility of the MSCP for use as long-term circulatory assist.

Plant-type L-asparaginases hydrolyze the side-chain amide bond of L-asparagine or its beta-peptides. They belong to the N-terminal nucleophile (Ntn) hydrolases and are synthesized as inactive precursor molecules. Activation occurs via the autoproteolytic release of two subunits, alpha and beta, the latter of which carries the nucleophile at its N-terminus. Crystallographic studies of plant-type asparaginases have focused on an Escherichia coli homologue (EcAIII), which has been crystallized in several crystal forms. Although they all belong to the same P2 1 2 1 2 1 space group with similar unit-cell parameters, they display different crystal-packing arrangements and thus should be classified as separate polymorphs. This variability stems mainly from different positions of the EcAIII molecules within the unit cell, although they also exhibit slight differences in orientation. The intermolecular interactions that trigger different crystal lattice formation are mediated by ions, which represent the most variable component of the crystallization conditions. This behaviour confirms recent observations that small molecules might promote protein crystal lattice formation.

A new Escherichia coli L-asparaginase belonging to the class of Ntn amidohydrolases has been crystallized using the vapour-diffusion method and PEG 4000 as the precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) (unit-cell parameters a = 50. 3, b = 77.6, c = 148.2 A) and diffract to 1.65 A resolution. The structure has been solved by molecular replacement using aspartylglucosaminidase from Flavobacterium meningosepticum as the search model. The asymmetric unit contains four protein chains composed into a dimer of alphabeta heterodimers, where the subunits alpha and beta are the product of autoproteolytic cleavage of the immature protein.

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.

BACKGROUND

Since the launch of Modernising Medical Careers, trainees are selected for a run-through training programme in a single surgical specialty. The surgical training bodies are currently considering the recommendations of the Tooke report as they review the policy for selection into surgical training in the UK. There is little information available on the factors involved in career choices amongst surgical trainees and this study aimed to address this issue.

METHODS

Trainees appointed to the Basic Surgical Training Programmes in the west and south-east of Scotland (1996-2006) were contacted by email and invited to participate in an online survey.

RESULTS

Of 467 trainees identified, valid email addresses were available for 299 of which 191 (64%) responded to the survey. One hundred and forty-nine (78%) trainees were still working in surgery but 38 (20%) had moved to a non-surgical specialty and 4 (2%) had left the medical profession. Of those who had obtained a NTN at the time of the survey (n = 138), 62 (45%) had a NTN in the specialty they chose at the start of the BST but 34 (25%) had changed to a different surgical specialty and 42 (30%) had left surgery altogether. For those still working in surgery, enjoyment of the specialty was the most important factor affecting career choice. Achieving an acceptable work/life balance was the most significant factor influencing trainees who left surgery.

CONCLUSIONS

The majority of trainees recruited to surgery at an early stage change specialty or leave surgery altogether. Both social and professional factors are important in career choices. The findings of this study support a period of core surgical training to provide flexibility prior to further training in a surgical specialty.

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.

Nephrotoxic nephritis (NTN) is characterized by a marked increase in glomerular eicosanoid synthesis, which appears to play an important role in the pathophysiology of this disease model. In this study, we investigated the biochemical and cellular basis of this metabolic change. By examining the enzymatic conversion of exogenous substrates by intact glomeruli, we found that cyclooxygenase, TX synthase, and 5-lipoxygenase activities increased 4-, 8-, and 100-fold, respectively, in acute NTN. PGH2-PGE2 isomerase and leukotriene A4 hydrolase activities did not change. The cellular basis of these changes was examined using dissociated glomerular cells in vitro and by depleting platelets in vivo. Dissociated glomerular cells from nephritic glomeruli (largely mesangial cells and leukocytes) exhibited an enhanced arachidonate metabolism similar to intact nephritic glomeruli. Depletion of neutrophils (PMNs) from these cell preparations by 90% commensurately decreased 5-lipoxygenase and cyclooxygenase activity but had little effect on TX synthase activity. The recovered PMN fraction, however, did exhibit TX synthase activity. Immunocytochemical analysis of dissociated cells using an antiplatelet antibody demonstrated the presence of platelets, both adherent to cells and noncell associated. Depletion of platelets in vivo using this antibody substantially attenuated the increase in glomerular eicosanoid synthesis that accompanied NTN. Platelet depletion also decreased the influx of PMNs into the glomerulus by 50%. These data show that PMNs and platelets colocalize to the glomerulus in acute NTN and are coordinately essential to the increase in glomerular arachidonate metabolism.

BACKGROUND

Genetically engineered neural stem cell (NSC) lines are promising vectors for the treatment of neurodegenerative diseases, particularly Parkinson's disease (PD). Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family, has been demonstrated to act specifically on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for PD. In our previous work, we demonstrated that NTN-overexpressing c17.2 NSCs exerted dopaminergic neuroprotection in a rat model of PD. In this study, we transplanted NTN-c17.2 into the striatum of the 6-hydroxydopamine (6-OHDA) PD model to further determine the regenerative effect of NTN-c17.2 on the rat models of PD.

RESULTS

After intrastriatal grafting, NTN-c17.2 cells differentiated and gradually downregulated nestin expression, while the grafts stably overexpressed NTN. Further, an observation of rotational behavior and the contents of neurotransmitters tested by high-performance liquid chromatography showed that the regenerative effect of the NTN-c17.2 group was significantly better than that of the Mock-c17.2 group, and the regenerative effect of the Mock-c17.2 group was better than that of the PBS group. Further research through reverse-transcriptase polymerase chain reaction assays and in vivo histology revealed that the regenerative effect of Mock-c17.2 and NTN-c17.2 cell grafts may be attributed to the ability of NSCs to produce neurotrophic factors and differentiate into tyrosine hydroxylase-positive cells.

CONCLUSIONS

The transplantation of NTN-c17.2 can exert neuroregenerative effects in the rat model of PD, and the delivery of NTN by NSCs may constitute a very useful strategy in the treatment of PD.

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto-oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto-oncogene. In introns 2 and 17 of the RET proto-oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRalpha, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed.

Potato tuber necrotic ringspot disease (PTNRD) is a damaging disease of potatoes, causing unsightly necrotic rings on the surface of tubers. The causal agent is thought to be tuber necrotic isolates of Potato virus Y, known as PVY(NTN). The disease spoils tubers for processing and table use, and the lack of a diagnostic method makes control especially difficult. The development of an RT-PCR assay for the reliable detection of PVY(NTN) and discrimination of all the main strains of PVY (PVY(O), PVY(N) and PVY(C)) is described. An assay was developed, exploiting a recombination site in the coat protein of PVY(NTN), allowing more reliable diagnosis of these isolates. Although the conserved nucleotide differences observed between the strains was very small, competitive PCR and mutagenically separated PCR were both employed in the development of a robust assay. The assay was found to be more reliable than the most commonly used RT-PCR method, and should prove to be an important tool in the confirmation of symptoms and for the detection of PVY(NTN) in symptomless tissue, in disease surveys and seed health schemes.

The reports of nontuberculous mycobacteria (NTM) associated with extrapulmonary diseases are increasing in tertiary care hospitals. Despite a significant increase in knowledge about NTM infections, they still represent a diagnostic and therapeutic challenge. The aim of this study is to know the prevalence of NTN among extrapulmonary tuberculosis cases in tertiary care centers in Northern India. A total of 227 culture positive isolates from 756 cases were tested for niacin production and catalase assay. BIO-LINE SD Ag MPT64 TB test and final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType Mycobacterium CM/AS assay. 71 cases (9.3%) were positive for AFB by ZN staining and 227 cases (30.1%) were positive for mycobacteria by culture. Niacin production and catalase activity were negative in 62/227 (27.4%) strains and after using a panel of different biochemicals and final confirmation by GenoType Mycobacterium CM assay. Out of 227 cultures tested, 165 (72.6%) strains were confirmed as M. tuberculosis complex, and 62 (27.4%) were confirmed as NTM. The most common NTM species identified were M. fortuitum 17 (27.5%) and M. intracellulare 13 (20.9%). The rapid identification of NTM species may help in targeted therapy and management of the diseases.

The effect of viral infection on the regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in Nicotiana tabacum L. leaves was studied. PEPC activity was 3 times higher in infected plant leaves compared to healthy plants. Activity of plant PEPC can be regulated, e.g., by de novo synthesis or reversible phosphorylation. The reason for the increase of PEPC activity as a consequence of PVY(NTN) infection was studied. The amount of PEPC determined by Western blot analysis or by relative estimation of PEPC mRNA by real-time PCR did not differ in control and PVY(NTN)-infected plants. Changes in posttranslational modification of PEPC by phosphorylation were evaluated by comparing activity of the native and the dephosphorylated enzyme. The infected plants were characterized by a higher decrease of the enzyme activity after its dephosphorylation, which indicated a higher phosphorylation level. Immunochemical detection of phosphoproteins by Western blot analysis showed a more intensive band corresponding to PEPC from the infected material. This strengthens the hypothesis of an infection-related phosphorylation, which could be part of the plant's response to pathogen attack. The physiological implications of the increase in PEPC activity during PVY(NTN) infection are discussed.

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVY(N)) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVY(NTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars 'Norchip' and 'Ranger Russet' developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of 'Russet Burbank'. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVY(N)-605 (98%) and with an Eu-PVY(NTN)-H (96%). However, when individual mature proteins were compared, much lower identities (86.5-94%) were found between Tu 660 and PVY9(NTN))-H compared to 98-99.5% between Tu 660 and PVY(N)-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVY(NTN)-H is a hybrid molecule of the genomic RNA recombination of PVY(O) and Eu-PVY(N) as shown by Glais et al. (Arch Virol 147, 363-378), whereas NA-PVY(NTN) Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVY(N) by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVY(NTN) Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.

OBJECTIVE

This prospective, randomized and blinded study compared the performance of a new needle-through-needle (NTN) kit (Epistar; Medimex, Germany) with the double-space technique for providing combined spinal epidural anaesthesia during Caesarean section.

METHODS

Following local Ethics Committee approval and patient consent, 200 females were randomized to receive combined spinal epidural anaesthesia by the double-space (n = 100) or NTN (n = 100) technique. The frequency with which the intrathecal component could achieve a T5 block to touch for Caesarean section without the need for epidural augmentation or an alternative technique was determined. The time from start of procedure to achieving a block height to T5 was recorded. Pain and backache at insertion, and at 24 h follow-up were recorded using a visual analogue scale. To remove any bias due to posture, 50% of each group were further randomized to receive their block in the sitting or in the left lateral position. To evaluate improvement of performance over time the success in the first 100 study patients were compared to the success in the second 100.

RESULTS

A successful block to T5 with the double-space and NTN techniques were 80 vs. 54, odds ratio 0.29. Failure to enter the intrathecal space once the epidural space had been located occurred in 29 patients in the NTN group. Time to readiness for surgery was 15 min (95% confidence interval (CI): 12.7-17.4) and 12.9 min (95% CI: 11.5-14.3) for the double-space and NTN techniques, respectively. The median (interquartile range) visual analogue scores for discomfort at insertion were 30 (12.5-51.5) and 32 (12.75-60) and for postoperative backache 0 (0-10) and 0 (0-10.75) in the double-space and NTN groups, respectively. The number of epidural augmentations was similar in both the groups and posture made no difference. There was a tendency to increased success in the second half of the study.

CONCLUSIONS

The double-space technique had a greater success rate than the NTN technique.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related neurotrophic factors that have both been shown to prevent the degeneration of dopaminergic neurons in vitro and in vivo. NTN and GDNF are thought to bind with different affinities to the GDNF family receptor alpha-2 (GFRalpha2), and can activate the same multi-component receptor system consisting of GFRalpha2, receptor tyrosine kinase Ret (RET) and NCAM. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that regulate gene expression through translational repression or RNA degradation. miRNAs have diverse functions, including regulating differentiation, proliferation and apoptosis in several organisms. It is currently unknown whether GDNF and NTN regulate the expression of miRNAs through activation of the same multi-component receptor system. Using quantitative real-time PCR, we measured the expression of some miRNA precursors in human BE(2)-C cells that express GFRalpha2 but not GFRalpha1. GDNF and NTN differentially regulate the expression of distinct miRNA precursors through the activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2). This study showed that the expression of distinct miRNA precursors is differentially regulated by specific ligands through the activation of GFRalpha2.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Nephrotoxic nephritis (NTN) is characterized by an influx of leukocytes into the glomerulus, with accompanying glomerular dysfunction and a marked increase in glomerular eicosanoid production. We examined the relationship between the glomerular inflammatory cell infiltrate and the concomitant metabolic/functional changes in this model of renal disease using a combination of in vivo immunologic strategies to both decrease [X-irradiation and cobra venom factor (CVF)] and increase (preimmunization with rabbit immunoglobulin G or accelerated NTN) the inflammatory cell infiltrate. With the use of these manipulations, a close correlation between glomerular leukocytes and the proteinuria of NTN was observed. The ablative strategies (X-irradiation and CVF) also attenuated the increase in leukotriene B4 (LTB4) generation seen with NTN and virtually completely prevented the increase in thromboxane B2 (TxB2) production (basal and angiotensin II elicited). Accelerated NTN, in contrast, increased and prolonged the rise in glomerular LTB4 production and exacerbated the increase in TxB2 production. Glomerular prostaglandin E2 production was not altered by the induction of nephritis nor any of the aforementioned immunologic manipulations. Regression analysis established that glomerular TxB2 production correlated significantly with the leukocyte influx at both 3 and 24 h. Glomerular LTB4 production correlated only with the presence of leukocytes at 3 h. Both glomerular TxB2 and LTB4 production were closely correlated with the renal dysfunction as assessed by proteinuria. These data suggest that leukocytes play a direct critical role in both the functional and metabolic alterations that occur in the setting of NTN. They further imply that leukocytes are crucial to the observed increase in glomerular eicosanoid production.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFR alpha 1 (considered to be the favored coreceptor for GDNF) or GFR alpha 2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2.

The Potato virus Y (PVY) cDNA full-length clone created by Jakab et al. [Jakab, G., Droz, E., Brigneti, G., Baulcombe, D., Malnoë, P., 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J. Gen. Virol. 78, 3141-3145] was stabilized by inserting three introns into putatively toxic genes. Using this clone, hybrid viruses were constructed by in vitro recombination. The PVY-N/NTN and PVY-N/O chimeras carried the 3' end of NIb, the whole CP and 3'UTR region of PVY(NTN) and PVY(O), respectively, in a PVY(N) genetic background. The clones proved to be stable after several passages by re-sequencing the exchanged region. Both hybrid viruses showed reduced infectivity in particle bombardment experiments, but they were suitable for further mechanical plant inoculation. In five of the six host plant species, inoculated with the two chimeras and three parental strains, the chimeras produced similar symptoms to those of PVY(N). By contrast, Physalis floridana reacted with different pattern of symptoms. In this species, the symptoms caused by the N/O hybrid were similar to those of the 3'NIb-CP-donating PVY(O) strain, and not to those of the background (PVY(N)). The results suggest that symptom determinants may be different even between strains of the same virus species in a particular host.

The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVYN) and potato tuber necrotic group (PVY(NTN)) contain segments of the PVYO and the PVY(N) genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVY(NTN) at sites HC/Pro-P3, 6K2-NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVYN isolates (termed PVY(N:O)). Protocols for specific differentiation of the recombinant PVY(NTN) and PVY(N:O) from the non-recombinant PVYN are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641bp from the recombinant PVY(NTN) and PVY(N:O). The second and third primer pairs exclusively amplified fragments of 448 and 290bp, respectively from the recombinant PVY(NTN). In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290bp) were amplified exclusively from the recombinant PVY(NTN), while only one fragment (641bp) was amplified from the PVY(N:O) isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVYO and North American (NA)-PVY(N/NTN). For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity.

Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

We studied the relation between immunopathology and progressive renal failure after nephrotoxic nephritis (NTN) in rats. Thirty days after induction of nephritis by injection of rabbit anti-rat nephrotoxic serum, pairs of kidneys from 13 nephritic rats were transplanted into separate syngeneic recipients, one of whom had been pre-immunized with rabbit immunoglobulin G (IgG) whilst the other was naive. Progression to renal failure of the transplanted nephritic kidney was studied after removal of the recipient's own kidneys; results from right and left kidney from a single donor in pre-immunized and naive recipients were compared. There were substantial differences in autologous anti-rabbit IgG titres in naive and preimmunized recipients; despite this pairs of kidneys from the same donor had almost identical courses as assessed by proteinuria, serum creatinine and graft survival. There was substantial variation in survival of kidneys from different donors. But there were very strong correlations of graft survival with proteinuria (r = 0.97, t = 4.443, P less than 0.001) and reciprocal serum creatinine (r = 0.95, t = 4.32, P less than 0.001) in donors shortly before transplantation. We conclude that autologous antibody titres did not influence the progression to renal failure after nephrotoxic nephritis. The rate of progression was already determined at the time of transplantation.

The RET receptor tyrosine kinase was first identified in a screen for human oncogenes and has subsequently been linked to several human syndromes: Hirschprung's disease, multiple endocrine neoplasia types 2A and 2B and familial thyroid carcinoma. Interestingly, all of the tissues affected by mutations in RET are derived from the neural crest during development. RET transduces a signal following activation by ligands of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophins which currently comprises GDNF, neuturin (NTN), artemin (ART) and persephin (PSP). To activate RET they form a tripartite complex with RET and a member of a family of four extracellular, GPI-linked alpha receptors (GFR alpha 1-4). Specificity is achieved by each GFR alpha binding only one member of the GDNF family with high affinity. Current evidence indicates that signal transduction by RET activates several second messenger systems including the PLC gamma, Ras, JNK and inositol phosphate pathways. Targeted mutagenesis in transgenic mice has shown that Ret, GFR alpha 1 and GDNF are required for multiple developmental events including development of the enteric nervous system (ENS) affected in Hirschsprung's disease. We describe experiments in chick neural crest cells which provide evidence for the normal function of RET and the basis of the defect in Hirschsprung's disease.

BACKGROUND

Diabetic peripheral neurovascular diseases (DPNVs) are complex, lacking effective treatment. Autologous/allogeneic transplantation of adipose-derived stem cells (ADSCs) is a promising strategy for DPNVs. Nonetheless, the transplanted ADSCs demonstrate unsatisfying viability, migration, adhesion, and differentiation in vivo, which reduce the treatment efficiency. Netrin-1 secreted as an axon guidance molecule and served as an angiogenic factor, demonstrating its ability in enhancing cell proliferation, migration, adhesion, and neovascularization.

METHODS

ADSCs acquired from adipose tissue were modified by Netrin-1 gene (NTN-1) using the adenovirus method (N-ADSCs) and proliferation, migration, adhesion, and apoptosis examined under high-glucose condition. The sciatic denervated mice (db/db) with type 2 diabetes mellitus (T2DM) were transplanted with N-ADSCs and treatment efficiency assessed based on the laser Doppler perfusion index, immunofluorescence, and histopathological assay. Also, the molecular mechanisms underlying Netrin-1-mediated proliferation, migration, adhesion, differentiation, proangiogenic capacity, and apoptosis of ADSCs were explored.

RESULTS

N-ADSCs improved the proliferation, migration, and adhesion and inhibited the apoptosis of ADSCs in vitro in the condition of high glucose. The N-ADSCs group demonstrated an elevated laser Doppler perfusion index in the ADSCs and control groups. N-ADSCs analyzed by immunofluorescence and histopathological staining demonstrated the distribution of the cells in the injected limb muscles, indicating chronic ischemia; capillaries and endothelium were formed by differentiation of N-ADSCs. The N-ADSCs group showed a significantly high density of the microvessels than the ADSCs group. The upregulation of AKT/PI3K/eNOS/P-38/NF-κB signaling pathways and secretion of multiple growth factors might explain the positive effects of Netrin-1 on ADSCs.

CONCLUSIONS

The overexpression of Netrin-1 in ADSCs improves proliferation, migration, and treatment effect in type 2 diabetic mice with sciatic denervation, which directs the clinical treatment of patients with DPNVs.