The purpose of this study was to evaluate the effect of combinations of blood pressure and glucose tolerance status on cardiovascular and all-cause mortality. A total of 7619 participants aged ⩾30 years old were stratified to nine categories as follows: (1) normotension (NTN) and normal glucose tolerance (NGT) (reference group), (2) NTN and pre-diabetes mellitus (pre-DM), (3) NTN and DM, (4) pre-hypertension (pre-HTN) and NGT, (5) pre-HTN and pre-DM, (6) pre-HTN and DM, (7) HTN and NGT, (8) HTN and pre-DM and (9) HTN and DM. Cox proportional hazards were applied to calculate the multivariate hazard ratios (HRs) of different groups for outcomes. For all-cause mortality outcomes, prevalent cardiovascular disease (CVD) was also adjusted. In a median follow-up of 11.3 years, 696 CVD and 412 all-cause mortality events occurred. Among the population free from CVD at baseline (n=7249), presence of HTN was associated with increased risk of CVD, regardless of glucose tolerance status with HRs of 1.97 (95% confidence interval (CI), 1.49-2.61), 2.25 (1.68-3.02) and 3.16 (2.28-4.37) for phenotypes of HTN and NGT, HTN and pre-DM and HTN and DM for CVD, respectively; corresponding HRs for all-cause mortality were 1.65 (95% CI, 1.15-2.37), 1.69 (1.15-2.49) and 2.73 (1.80-4.14), respectively. Phenotypes of NTN and pre-DM (1.48; 1.03-2.14) and NTN and DM (2.04; 1.06-3.92) were also associated with CVD and all-cause mortality, respectively. HTN was significantly associated with CVD/mortality events, regardless of glucose tolerance status. Blood pressure <120/80 mm Hg among pre-diabetic/diabetic population, not on antihypertensive medications, was generally associated with worse outcomes.

Indirect measurement of the flow rate of a centrifugal blood pump using the driving motor current was studied. The pump flow rate can be expressed as a function of the motor current under a given motor speed in the absence of energy loss resulting from uncertain mechanical contact friction. The magnetically suspended centrifugal blood pump (MSCP), developed by the collaboration of Kyoto University and NTN Inc., was suitable for the application of this measuring method because the impeller is suspended magnetically inside the pump housing without any mechanical contact. The effect of fluid viscosity on the pump performance was investigated in detail, and it was possible to estimate the pump flow rate and the pressure difference through the pump (from inlet port to outlet port) accurately by monitoring the motor current and speed when the kinematic viscosity of working fluids was known. The kinematic viscosity of working fluids can also be measured with the MSCP. The motor current and motor speed were monitored in a chronic animal experiment, and the estimated flow rate and pressure difference showed good correlation with directly measured data.

The β-aminopeptidase BapA from Sphingosinicella xenopeptidilytica belongs to the N-terminal nucleophile (Ntn) hydrolases of the DmpA-like family and has the unprecedented property of cleaving N-terminal β-amino acid residues from peptides. We determined the crystal structures of the native (αβ)₄ heterooctamer and of the 153 kDa precursor homotetramer at a resolution of 1.45 and 1.8 Å, respectively. These structures together with mutational analyses strongly support mechanisms for autoproteolysis and catalysis that involve residues Ser250, Ser288, and Glu290. The autoproteolytic mechanism is different from the one so far described for Ntn hydrolases. The structures together with functional data also provide insight into the discriminating features of the active site cleft that determine substrate specificity.

We studied the behavior of myogenic progenitors from donor desmin(+/-) LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of beta-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.

The occurrence of the glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) was examined by immunohistochemistry in the normal human brainstem at pre-, perinatal and adult age. Immunolabelled neurons were unevenly distributed and each trophin had a consistent distribution pattern. As a rule, the NTN antiserum produced the most abundant and diffuse tissue labelling, whereas the lowest density of positive elements was observed after ART immunostaining. Labelling for NTN, PSP, and ART occurred at all examined ages. For each trophin, neuronal perikarya were observed within sensory and motor nuclei of cranial nerves, dorsal column nuclei, olivary nuclear complex, reticular formation, pontine nuclei, locus caeruleus, raphe nuclei, substantia nigra, and quadrigeminal plate. Nerve fibers occurred within gracile and cuneate fasciculi, trigeminal spinal tract and nucleus, oculomotor and facial nerves, solitary tract, vestibular nerve, medial longitudinal fasciculus, medial and lateral lemnisci, and inferior and superior cerebellar peduncles. Age changes were detected in the distribution pattern for each trophin. On the whole, in the grey matter, labelled perikarya were more frequently observed in pre- and perinatal than in adult specimens; on the other hand, in discrete regions, nerve fibers and terminals were abundant and showed a definite arrangement only in adult tissue; finally, distinct fiber systems in the white matter were immunolabelled only at pre- and perinatal ages. The results support the concept of a trophic involvement of NTN, PSP, and ART in the development, functional activity and maintenance of a variety of human brainstem neuronal systems.

The objective of these experiments was to determine whether living and training in moderate hypoxia (MHx) confers an advantage on maximal normoxic exercise capacity compared with living and training in normoxia. Rats were acclimatized to and trained in MHx [inspired PO2 (PI(O2)) = 110 Torr] for 10 wk (HTH). Rats living in normoxia trained under normoxic conditions (NTN) at the same absolute work rate: 30 m/min on a 10 degrees incline, 1 h/day, 5 days/wk. At the end of training, rats exercised maximally in normoxia. Training increased maximal O2 consumption (VO2 max) in NTN and HTH above normoxic (NS) and hypoxic (HS) sedentary controls. However, VO2 max and O2 transport variables were not significantly different between NTN and HTH: VO2 max 86.6 +/- 1.5 vs. 86.8 +/- 1.1 ml x min(-1) x kg(-1); maximal cardiac output 456 +/- 7 vs. 443 +/- 12 ml x min(-1) x kg(-1); tissue blood O2 delivery (cardiac output x arterial O2 content) 95 +/- 2 vs. 96 +/- 2 ml x min(-1) x kg(-1); and O2 extraction ratio (arteriovenous O2 content difference/arterial O2 content) 0.91 +/- 0.01 vs. 0.90 +/- 0.01. Mean pulmonary arterial pressure (Ppa, mmHg) was significantly higher in HS vs. NS (P < 0.05) at rest (24.5 +/- 0.8 vs. 18.1 +/- 0.8) and during maximal exercise (32.0 +/- 0.9 vs. 23.8 +/- 0.6). Training in MHx significantly attenuated the degree of pulmonary hypertension, with Ppa being significantly lower at rest (19.3 +/- 0.8) and during maximal exercise (29.2 +/- 0.5) in HTH vs. HS. These data indicate that, despite maintaining equal absolute training intensity levels, acclimatization to and training in MHx does not confer significant advantages over normoxic training. On the other hand, the pulmonary hypertension associated with acclimatization to hypoxia is reduced with hypoxic exercise training.

The role of DC-SIGN in tubulointerstitial lesions (TILs) and the effect of anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) were investigated in rat nephrotoxic nephritis (NTN). On Day 4, immature DC-SIGN+DCs infiltrated into renal tubulointerstitium and matured by Day 14, showing increased migratory capacity and ability to induce T cell proliferation. The distribution of DC-SIGN+ DC significantly correlated with crescent formation, TIL severity, and changes in renal function. RANTES and TNF-alpha mRNA were continuously up-regulated from Day 4, while IL-10 mRNA was down-regulated after a marked increase on Day 4. Expression of IFN-gamma and IL-4 mRNA increased on Day 14 due to DC maturation. PsL-EGFmAb suppressed DC maturation, migration and ability to activate T cells. It also down-regulated TNF-alpha and up-regulated IL-10, resulting in a Th1/Th2 bias. The number of crescents decreased and TILs and renal function improved. These results suggest that DC-SIGN mediates DC tubulointerstitial infiltration and is an important regulator of local immune reactions and TILs. PsL-EGFmAb inhibited DC migration, maturation and function by targeting DC-SIGN, and may therefore be a potential treatment for NTN.

The origin of proliferating cells in glomeruli and Bowman's capsules during nephrotoxic nephritis (NTN) in rabbits was studied, using unilateral renal irradiation to suppress glomerular cell division. Left (LI) kidneys received 2400-12,000 rad on Days 1-5 after i.v. nephrotoxic serum (NTS) 1 or 1.5 ml/kg body wt. Renal histology, cell labelling 1 h after a pulse of tritiated thymidine (H3T) (0.5 mCi/kg), and mitotic rate were compared in right non-irradiated (RO) kidneys and LI kidneys. Differences in H3T uptake were not significantly suppressed (P less than 0.05) by 4800 rad whereas mitoses in glomeruli and Bowman's capsule were not. Glomerular hypercellularity and crescents were equivalent in LI given 4800 rad and in RO kidneys, but fibrin deposition was increased 2-fold in LI kidneys. In control rabbits (no NTS) LI kidneys showed no histological changes. The results suggest that most dividing cells detected in glomeruli and Bowman's space in NTN are monocytes, and that these cells form a large component of crescents.

Computational fluid dynamic simulations of the Kyoto-NTN magnetically suspended centrifugal blood pump with 16 forward-bending blades (16FB), 16 straight blades (16SB), and eight backward-bending blades (8BB) impellers were performed in this study. Commercial CFD software package FLUENT were used as the solver. The purpose of this study is to find out how the impeller blade profiles affect the inner flow and the performance of the centrifugal blood pump. The simulations were carried out with the same impeller rotating speed of 2,000 rpm and pump flow rate of 5 L/min to compare the three pump models. It was found that the 16SB impeller can produce higher pressure head than the 16FB and 8BB impellers under the same impeller rotating speed and pump flow rate. The flow particle tracing was carried out to estimate the blood damage level caused by the three different impeller profiles. It was found that the 16FB and 8BB models have caused the highest and lowest blood damage, respectively. The 16SB is recommended among the three pumps because it can generate the highest pressure head and induce mild blood damage index, although it was higher than that of the 8BB model.

Neurotrophins such as nerve growth factor and brain-derived neurotrophic factor have been described to be involved in the pathogenesis of asthma. Neurturin (NTN), another neurotrophin from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN(-/-)) mice. NTN is structurally similar to TGF-β, a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway inflammation using NTN(-/-) mice. The bronchial inflammatory response of OVA-sensitized NTN(-/-) mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN(-/-) mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly enhanced in NTN(-/-) mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before challenge with OVA partially rescues the phenotype of NTN(-/-) mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma.

: We generalize some current approaches for RNA tree alignment, which are traditionally confined to ordered rooted mappings, to also consider unordered unrooted mappings. We define the Homeomorphic Subtree Alignment problem (HSA), and present a new algorithm which applies to several modes, combining global or local, ordered or unordered, and rooted or unrooted tree alignments. Our algorithm generalizes previous algorithms that either solved the problem in an asymmetric manner, or were restricted to the rooted and/or ordered cases. Focusing here on the most general unrooted unordered case, we show that for input trees T and S, our algorithm has an O(nTnS + min(dT,dS)LTLS) time complexity, where nT,LT and dT are the number of nodes, the number of leaves, and the maximum node degree in T, respectively (satisfying dT ≤ LT ≤ nT), and similarly for nS,LS and dS with respect to the tree S. This improves the time complexity of previous algorithms for less general variants of the problem.In order to obtain this time bound for HSA, we developed new algorithms for a generalized variant of the Min-Cost Bipartite Matching problem (MCM), as well as to two derivatives of this problem, entitled All-Cavity-MCM and All-Pairs-Cavity-MCM. For two input sets of size n and m, where n ≤ m, MCM and both its cavity derivatives are solved in O(n3 + nm) time, without the usage of priority queues (e.g. Fibonacci heaps) or other complex data structures. This gives the first cubic time algorithm for All-Pairs-Cavity-MCM, and improves the running times of MCM and All-Cavity-MCM problems in the unbalanced case where n ≪ m.We implemented the algorithm (in all modes mentioned above) as a graphical software tool which computes and displays similarities between secondary structures of RNA given as input, and employed it to a preliminary experiment in which we ran all-against-all inter-family pairwise alignments of RNAse P and Hammerhead RNA family members, exposing new similarities which could not be detected by the traditional rooted ordered alignment approaches. The results demonstrate that our approach can be used to expose structural similarity between some RNAs with higher sensitivity than the traditional rooted ordered alignment approaches. Source code and web-interface for our tool can be found in http://www.cs.bgu.ac.il/\~negevcb/FRUUT.

Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family, has substantial effects on normal and lesioned nigrostriatal dopamine systems. However, its ability to protect against toxin-induced loss of striatal dopamine release has not been previously reported. The goal of the present study was to determine if NTN could protect against 6-hydroxydopamine (6-OHDA)-induced reductions in striatal dopamine overflow and tissue levels of dopamine and to compare the effects of NTN with those of GDNF. Male Fischer-344 rats were given a single injection of vehicle, or 5 microg NTN or GDNF, into the right striatum. The following day the animals were given a single injection of 12 microg 6-OHDA into the striatum at the same site where the trophic factor was injected. Microdialysis experiments conducted three weeks later indicated that the 6-OHDA decreased basal levels of dopamine and metabolites in the lesioned striatum compared to the contralateral striatum, and NTN was able to partially protect against the 6-OHDA-induced reductions. Injection of NTN one day prior to 6-OHDA also led to significant protection against loss of both potassium- and amphetamine-evoked overflow of dopamine. The NTN treatments partially protected against 6-OHDA-induced reductions in striatal tissue levels of dopamine and completely protected against loss of nigral dopamine content. The protective effects of NTN were similar in magnitude to those of GDNF. These results support that within the experimental parameters used in this study, NTN is as effective as GDNF in protecting against the dopamine-depleting effects of intrastriatal 6-OHDA.

BACKGROUND

The lysosomal 66.3 kDa protein from mouse is a soluble, mannose 6-phosphate containing protein of so far unknown function. It is synthesized as a glycosylated 75 kDa precursor that undergoes limited proteolysis leading to a 28 kDa N- and a 40 kDa C-terminal fragment.

RESULTS

In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core alphabetabetaalpha sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily.

CONCLUSIONS

Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.

Previous studies have shown that platelet-activating factor (PAF) receptor blocking has a protective effect on rabbit nephrotoxic nephritis (NTN). We examined whether arachidonic acid (AA) metabolism is altered in NTN and whether a PAF receptor antagonist has any influence on such changes. Rabbits injected with anti-glomerular basement membrane antiserum in the heterologous phase had a markedly increased glomerular thromboxane B2 (TxB2) production level, whereas no changes have been detected in glomerular 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2). During the autologous phase of the disease, the glomerular TxB2 level was even higher than in the heterologous phase. The level of 6-keto-PGF1 alpha was significantly lower than normal, and the level of PGE2 was unchanged in respect to the basal values. The use of L-652,731 (a specific PAF receptor antagonist) reversed the abnormal generation of AA metabolites at glomerular level both in the heterologous and autologous phase of the disease. The effect of L-652,731 on AA metabolism is likely to be an indirect result of the PAF receptor blocking, because L-652,731 given to normal rabbits had no direct effect on glomerular AA metabolism. To assess whether the beneficial effect of L-652,731 in NTN is at least in part mediated by its capability of suppressing the excessive intrarenal synthesis of thromboxane A2 (TxA2), we compared the effect of L-652,731 with that of a selective TxA2-synthase inhibitor (FCE-22178). FCE-22178 ameliorated the morphologic expression of rabbit NTN and reduced function deterioration. The protective effect of L-652,731 on proteinuria in the autologous phase and on glomerular filtration rate in both phases was superior to that of FCE-22178. We conclude that an excessive intraglomerular synthesis of TxA2 occurs in rabbit NTN that can play a role in renal function deterioration. Both a specific PAF receptor antagonist and a TxA2-synthase inhibitor reduced the exaggerated TxA2 synthesis and favorably influenced the evolution of the disease.

During moderate cold exposure, cardiovascular-related morbidity and mortality increase disproportionately in hypertensive adults (HTN); however, the mechanisms underlying this association are not well defined. We hypothesized that whole body cold stress would evoke exaggerated increases in blood pressure (BP) and muscle sympathetic nerve activity (MSNA) in HTN compared with normotensive adults (NTN) and that sympathetic baroreflex function would be altered during cooling in HTN. MSNA (peroneal microneurography) and beat-to-beat BP (Finometer) were measured continuously in 10 NTN (6 men/4 women; age 53 ± 3 yr; resting BP 125 ± 3/79 ± 1 mmHg) and 13 HTN (7 men/6 women; age 58 ± 2 yr; resting BP 146 ± 5/88 ± 2 mmHg) during whole body cooling-induced reductions in mean skin temperature (Tsk; water-perfused suit) from 34.0 to 30.5°C. During cooling, the increase in mean arterial pressure was greater in HTN (NTN: Δ6 ± 2 vs. HTN: Δ11 ± 1 mmHg; P = 0.02) and accompanied by exaggerated increases in MSNA (NTN: Δ8 ± 3 vs. HTN: Δ20 ± 3 bursts/100 heart beats; P < 0.01). The slope of the relation between MSNA and diastolic BP did not change during cooling in NTN (Tsk 34.0°C: -4.4 ± 0.8 vs. Tsk 30.5°C: -5.0 ± 0.3 bursts·100 heart beats-1·mmHg-1; P = 0.47) but increased in HTN (Tsk 34.0°C: -3.6 ± 0.4 vs. Tsk 30.5°C: -5.4 ± 0.4 bursts·100 heart beats)-1·mmHg-1; P = 0.02). These findings demonstrate that the cooling-induced increases in BP and MSNA are exaggerated in HTN. Furthermore, during cooling, sympathetic baroreflex sensitivity increases in HTN, but not NTN, presumably to allow for baroreflex-mediated buffering of excessive cooling-induced increases in BP. Collectively, these findings suggest that sympathetic function is altered during whole body cooling in hypertension. NEW & NOTEWORTHY These novel findings demonstrate that whole body cooling-induced reductions in mean skin temperature elicited greater increases in blood pressure and muscle sympathetic nerve activity in hypertensive adults. In addition, during moderate cold exposure, sympathetic baroreflex sensitivity increased in hypertensive, but not normotensive, adults.

BACKGROUND

Stem cell factor (SCF) has been implicated in many disease processes characterized by tissue remodelling and fibrosis. The growth factor (SCF) was evaluated in a rat model of nephrotoxic serum nephritis (NTN), characterized by early inflammation followed by later tissue fibrosis.

METHODS

NTN was induced in male Wistar Kyoto rats using rabbit anti-rat glomerular basement membrane antibodies. Animals were sacrificed at days 7, 15, 30 and 45 (n = 4-10 per group). Rats' kidneys were immunostained for ED1 as marker of inflammation, CD34, SCF, c-kit, mast cell tryptase and markers of fibrosis; collagens III and IV and alpha-SMA. Changes in SCF protein and mRNA content were evaluated by Western blotting and Northern blotting, respectively.

RESULTS

In the NTN kidney, levels of immunoreactive SCF and SCF receptor (c-kit) were significantly higher in glomerular, tubular and interstitial compartments. Mast cells were barely detectable in NTN and control rat sections. Double immunostaining showed the co-localization of SCF with alpha-SMA and of the SCF receptor with CD34 and ED1 positive cells. Immunostainable SCF protein in each of the 3 compartments, glomerular, tubular and interstitial, showed a positive linear correlation with serum creatinine, proteinuria, glomerulosclerosis score and interstitial fibrosis scores. Using multivariate analysis, immunostainable tubular SCF was a predictor of glomerular sclerosis and immunostainable glomerular SCF predicted tubular atrophy. Increased SCF immunostain was not a consequence of altered transcription as there was a fall in SCF mRNA determined by Northern blotting. Western blotting of NTN kidney homogenates revealed two bands for SCF, a 43-kDa band which decreased, and a 19-kDa band which increased throughout the study.

CONCLUSIONS

These results highlight the potential role of SCF and its receptor in the remodelling process of the NTN kidney. Upregulation of SCF may involve a translational mechanism, with the soluble SCF protein KL-S1 (19 kDa) being derived from the transmembrane SCF protein KL-1 (43 kD) by proteolytic cleavage. The immunohistochemical staining of few CD34+ cells in NTN kidneys warrants further evaluation of the nature of these cells in the context of the inflammatory as well as the fibrotic processes.

Potato virus Y (PVY) strains were originally defined by interactions with different resistance genes in standard potato cultivars. Five distinct strain groups are defined that cause local or systemic hypersensitive responses (HRs) in genetic background with a corresponding N gene: PVY(O), PVY(N), PVY(C), PVY(Z), and PVY(E). The nucleotide sequences of multiple isolates of PVY(O) and PVY(N) differ from each other by ≈8% along their genomes. Additionally, complete genome sequences of multiple recombinant isolates are composed of segments of parental PVY(O) and PVY(N) sequences. Here, we report that recombinant isolate PVY-L26 induces an HR in potato 'Maris Bard' carrying the putative Nz gene, and is not recognized by two other resistance genes, Nc and Ny(tbr). These genetic responses in potato, combined with the inability of PVY-L26 to induce vein necrosis in tobacco, clearly define it as an isolate from the PVY(Z) strain group and provide the first information on genome structure and sequence of PVY(Z). The genome of PVY-L26 displays typical features of European NTN-type isolates with three recombinant junctions (PVY(EU-NTN)), and the PVY-L26 is named PVY(Z)-NTN. Three typical PVY(NTN) isolates and two PVY(N) isolates, all inducing vein necrosis in tobacco, were compared with PVY-L26. One PVY(NTN) isolate elicited HR reactions in Maris Bard, similar to PVY-L26, while two induced a severe systemic HR-like reaction quite different from the quasi-symptomless reaction induced by two PVY(N) isolates. 'Yukon Gold' potato from North America produced HR against several PVY(NTN) isolates, including PVY-L26, but only late and limited systemic necrosis against one PVY(N) isolate. Consequently, according to symptoms in potato indicators, both PVY(Z) and PVY(NTN) isolates appeared biologically very close and clearly distinct from PVY(O) and PVY(N) strain groups.

The effect of biotic stress induced by viral infection (Potato virus Y, strain NTN and O) on NADP-malic enzyme (EC 1.1.1.40) in tobacco plants (Nicotiana tabacum L., cv. Petit Havana, SR1) was tested at the transcriptional, translational and activity level. The increase of enzyme activity in infected leaves was correlated with the increased amount of expressed protein and with mRNA of cytosolic NADP-ME isoform. Transcription of the chloroplastic enzyme was not influenced by viral infection. The increase of the enzyme activity was also detected in stems and roots of infected plants. The effect of viral infection induced by Potato virus Y, NTN strain, causing more severe symptoms, was compared with the effect induced by milder strain PVY(O). The observed increase in NADP-malic enzyme activity in all parts of the studied plants was higher in the case of PVY(NTN) strain than in the case of strain PVY(O). The relevance of NADP-malic enzyme in plants under stress conditions was discussed.

As part of a routine screening for the possible presence of the necrotic strains of potato virus Y affecting potatoes in Mexico, five PVY isolates were submitted to biological and molecular analysis. Considering their serological properties, two belong to the common strain (O) and three to the necrotic strain (N). All the isolates induced vein necrosis in Nicotiana tabacum. To characterize the isolates, 5' NTR and P1 genes were sequenced and compared with sequences from GenBank database. Nucleotide sequence similarity ranged from 47-100% in the 5' NTR and from 63-100% in the P1 coding region. The lowest amino acid similarity between sequences of P1 gene was 55%. In phylogenetic trees of 5'NTR analysis, two PVY(O) Mexican isolates clustered with other PVY(O) isolates. In turn, the three PVY(N) isolates grouped with PVY(N-NTN) isolates. The phylogenetic analysis of P1 sequences (nucleotide and amino acid) showed two PVY(O) isolates grouping next to N-NTN cluster. A detailed analysis of the PVY(O) isolates showed two potential recombination regions in the P1 gene, in contrast to 5'NTR where no recombination was detected.

Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.

BACKGROUND

The role of miRNAs in familial breast cancer (fBC) is poorly investigated as also in the BRCA-like tumors. To identify a specific miRNA expression pattern which could allow a better fBC classification not only based on clinico-pathological and immunophenotypical parameters we analyzed miRNA profile in familial and sporadic samples. Moreover since BRCA1 tumors and sporadic triple negative (TN) breast tumors share similarities regarding clinical outcomes and some histological characteristics, we focused on TN and not TN cases.

METHODS

The sample set included fresh frozen tissue samples, including 39 female fBCs (19 BRCA-related and 20 BRCAX) and 12 male fBC (BRCAX). Moreover, we considered TN and non TN (NTN), 21 BRCA-related and 27 sporadic BCs. MiRNA profiling was performed through GeneChip miRNA v.1.0 Array (Affymetrix). ANOVA, hierarchical and consensus clustering analyses allowed identification of pattern of expression of miRNAs and pathway enrichment analysis, considering validated target genes, was carried out to achieve a deeper biological understanding.

RESULTS

ANOVA test led to the identification of 53 deregulated miRNAs; hierarchical and consensus clustering of female fBCs (fFBCs) and male fBCs (fMBCs) highlighted the presence of 3 sample clusters named FBC1, FBC2 and FBC3. We found a correlation between ER-status and the three sample clusters. The three clusters are distinct by a different expression of two clusters of miRNAs (CLU1 and CLU2), which resulted to be different in targeted pathways. In particular, CLU1 targets cellular pathways and CLU2 is involved in epigenetic activities. Considering TN and NTN BRCA-related and sporadic tumors, a hierarchical clustering identified two clusters of miRNAs, which were not so different from CLU1 and CLU2, both in miRNA content and targeted pathways.

CONCLUSIONS

Our results highlighted the importance of miRNA regulation to better clarify similarities and differences between familial and sporadic BC groups.

OBJECTIVE

Difficulties in obtaining properly preserved human cochlea have been a major obstacle to in vitro study of this deeply located and hard bone-fortressed hearing organ. Our study aimed at investigating GDNF family ligands (GFLs) and their receptors in the human cochleae that were surgically obtained during a transcochlear approach dealing with life-threatening, intra-cranial meningiomas.

METHODS

The specimens were properly fixed with 4% paraformaldehyde in the operating room. By using immunohistochemical techniques, distribution of GDNF, Neurturin (NTN, one member of GFLs), as well as cRet, GFRα-1 and GFRα-2 receptors in the human cochleae was investigated. Five cochleae from five adult patients were processed for the study. The patients had normal hearing threshold before operation.

RESULTS

cRet receptor immunoreactivity was seen in the spiral ganglion neurons, mainly inside the cell bodies but rarely in the nerve fibers and not in the organ of Corti. Immunolabeling for GFRα-1 and GFRα-2 receptors was identified mainly in the cell bodies of the spiral neurons than in the nerve fibers. In the organ of Corti, GFRα-1 immunostaining could be demonstrated in the Deiters' cells, Hensen cells, inner pillar cells, and weakly in the inner hair cells but not in the outer hair cells; no structures in the organ of Corti were labeled with GFRα-2 receptor antibody. NTN immunostaining was found in the supporting cells of organ of Corti, including Deiters' cells, Hensen cells as well as Claudius' cells. In the spiral ganglia, NTN immunostaining was seen in both the cell bodies and the nerve fibers of neurons. GDNF immunoreactivity was not revealed in human cochlea.

CONCLUSIONS

Surgically obtained human cochleae were properly fixed and underwent immunohistochemical investigation of neurotrophic elements. NTN and its receptors discovered in current study can be responsible for the unique neuronal survival properties in human spiral ganglion (hSG); a prerequisite for the function of cochlear implants.

Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of ganglion cells in the nerve plexuses of the lower digestive tract. Although mutations in eight different genes (EDNRB, EDN3, ECE1, SOX10, RET, GDNF, NTN, SIP1) have been identified in affected individuals, it is now clear that RET and EDNRB are the primary genes implicated in the etiology of HSCR. All eight genes are involved in the early development of the enteric nervous system, and most act through two distinct biochemical pathways mediated by RET and EDNRB. Mutations in RET and EDNRB account for up to 50% and 5% of HSCR cases in the general population, respectively. Interaction between these two signaling pathways could modify RET expression and, therefore, HSCR phenotype. Here, we report the case of a 1-year-old Taiwanese boy who presented with abdominal distension since birth and bilious vomiting after feeding. HSCR (short-segment type) was diagnosed based on X-ray, lower gastrointestinal series and biopsy findings. Mutation analysis revealed a heterozygous T>C missense mutation in exon 1 of the EDNRB gene, that substitutes the highly conserved cysteine-90 residue in the extracellular domain of the G protein-coupled receptor with an arginine residue (C90R). No RET gene mutation was detected in this patient.