Contrast-Enhanced Spectral Mammography (CESM) is a recently introduced mammographic method with characteristics particularly suitable for breast cancer radiomic analysis. This work aims to evaluate radiomic features for predicting histological outcome and two cancer molecular subtypes, namely Human Epidermal growth factor Receptor 2 (HER2)-positive and triple-negative. From 52 patients, 68 lesions were identified and confirmed on histological examination. Radiomic analysis was performed on regions of interest (ROIs) selected from both low-energy (LE) and ReCombined (RC) CESM images. Fourteen statistical features were extracted from each ROI. Expression of estrogen receptor (ER) was significantly correlated with variation coefficient and variation range calculated on both LE and RC images; progesterone receptor (PR) with skewness index calculated on LE images; and Ki67 with variation coefficient, variation range, entropy and relative smoothness indices calculated on RC images. HER2 was significantly associated with relative smoothness calculated on LE images, and grading tumor with variation coefficient, entropy and relative smoothness calculated on RC images. Encouraging results for differentiation between ER+/ER-, PR+/PR-, HER2+/HER2-, Ki67+/Ki67-, High-Grade/Low-Grade and TN/NTN were obtained. Specifically, the highest performances were obtained for discriminating HER2+/HER2- (90.87%), ER+/ER- (83.79%) and Ki67+/Ki67- (84.80%). Our results suggest an interesting role for radiomics in CESM to predict histological outcomes and particular tumors' molecular subtype.

Keywords: Her2 positive; breast cancer; correlation; histological outcome; prediction; radiomic analysis; triple negative.

Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson's disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury.

The role of IL-6 signaling in renal diseases remains controversial, with data describing both anti-inflammatory and proinflammatory effects. IL-6 can act via classic signaling, engaging its two membrane receptors gp130 and IL-6 receptor (IL-6R). Alternatively, IL-6 trans-signaling requires soluble IL-6R (sIL-6R) to act on IL-6R-negative cells that express gp130. Here, we characterize the role of both pathways in crescentic nephritis. Patients with crescentic nephritis had significantly elevated levels of IL-6 in both serum and urine. Similarly, nephrotoxic serum-induced nephritis (NTN) in BALB/c mice was associated with elevated serum IL-6 levels. Levels of serum sIL-6R and renal downstream signals of IL-6 (phosphorylated signal transducer and activator of transcription 3, suppressor of cytokine signaling 3) increased over time in this model. Simultaneous inhibition of both IL-6 signaling pathways using anti-IL-6 antibody did not have a significant impact on NTN severity. In contrast, specific inhibition of trans-signaling using recombinant sgp130Fc resulted in milder disease. Vice versa, specific activation of trans-signaling using a recombinant IL-6-sIL-6R fusion molecule (Hyper-IL-6) significantly aggravated NTN and led to increased systolic BP in NTN mice. This correlated with increased renal mRNA synthesis of the Th17 cell cytokine IL-17A and decreased synthesis of resistin-like alpha (RELMalpha)-encoding mRNA, a surrogate marker of lesion-mitigating M2 macrophage subtypes. Collectively, our data suggest a central role for IL-6 trans-signaling in crescentic nephritis and offer options for more effective and specific therapeutic interventions in the IL-6 system.

Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, because several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes, it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar K(m) value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we determined the structure of human l-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn hydrolases, this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state, we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage and the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased K(m) value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.

Understanding relationships between sequence, structure, and evolution is important for functional characterization of proteins. Here, we define a novel DOM-fold as a consensus structure of the domains in DmpA (L-aminopeptidase D-Ala-esterase/amidase), OAT (ornithine acetyltransferase), and MocoBD (molybdenum cofactor-binding domain), and discuss possible evolutionary scenarios of its origin. As shown by a comprehensive structure similarity search, DOM-fold distinguished by a two-layered beta/alpha architecture of a particular topology with unusual crossing loops is unique to those three protein families. DmpA and OAT are evolutionarily related as indicated by their sequence, structural, and functional similarities. Structural similarity between the DmpA/OAT superfamily and the MocoBD domains has not been reported before. Contrary to previous reports, we conclude that functional similarities between DmpA/OAT proteins and N-terminal nucleophile (Ntn) hydrolases are convergent and are unlikely to be inherited from a common ancestor.

A Seasonal Kendall Trend (SKT) test was applied to precipitation-weighted concentration data from 164 National Atmospheric Deposition Program National Trends Network (NADP/NTN) sites operational from 1985 to 2002. Analyses were performed on concentrations of ammonium, sulfate, nitrate, dissolved inorganic nitrogen (DIN, sum of nitrogen from nitrate and ammonium), and earth crustal cations (ECC, sum of calcium, magnesium, and potassium). Over the 18-year period, statistically significant (p< or =0.10) increases in ammonium concentrations occurred at 93 sites (58%), while just three sites had statistically significant ammonium decreases. Central and northern Midwestern states had the largest ammonium increases. The generally higher ammonium concentrations were accompanied by significant sulfate decreases (139 sites, 85%), and only one significant increase which occurred in Texas. In the west central United States there were significant nitrate increases (45 sites, 27%), while in the northeastern United States there were significant decreases (25 sites, 15%). Significant DIN decreases were observed in the northeastern United States (11 sites, 7%); elsewhere there were significant increases at 75 sites (46%). ECC decreased significantly at 65 sites (40%), predominantly in the central and southern United States, and increased at 11 sites (7%). The concentrations of sulfate, nitrate, and ammonium in precipitation have changed markedly over the time period studied. Such trends indicate changes in the mix of gases and particles scavenged by precipitation, possibly reflecting changes in emissions, atmospheric chemical transformations, and weather patterns.

Ret is the receptor for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN). Defects in this receptor underlie several genetic syndromes. The receptor is a transmembrane tyrosine kinase which transduces Ret-mediated signaling to a variety of signaling pathways, most notably the Ras signaling pathway and the phosphatidylinositol-3 kinase pathway. These pathways are activated through the interaction of adaptor proteins to tyrosine phosphorylated receptor. The ultimate biological effects, depending on the cell type, include morphological changes in the cytoskeleton, cell scattering, proliferation, and differentiation.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) sequences separated into at least two distinct lineages on the whole-genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVY(NTN) occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs to be minimised to prevent further such occurrences.

Hirschsprung's disease, affecting one in 5000 live newborns, is the most common cause of neonatal intestinal obstruction. The obstruction or, later in life, constipation arises from the lack of enteric ganglia in the hindgut, thus resulting in poor coordination of peristalsis. Mutations in Hirschsprung patients have so far been reported in five genes associated in two different receptor-ligand systems, RET-GDNF/NTN and EDNRB-EDN-3, and an additional gene with yet unknown precise function, SOX10. We report the results of single-stranded conformation polymorphism screening of the endothelin-3 gene in a Swedish population-based material of 66 sporadic and nine familial Hirschsprung's disease cases. We have found a novel heterozygous mutation in exon 2, c.262insG, in a patient with sporadic short segment Hirschsprung's disease without any Waardenburg features. This frameshift results in a premature stop two codons further on. Because this stop is introduced 5' of the biologically active protein, this mutation can hence be predicted to result in haplo-insufficiency.

BACKGROUND

Potato virus Y (PVY) is one of the most important plant viruses affecting potato production. The interactions between potato and PVY are complex and the outcome of the interactions depends on the potato genotype, the PVY strain, and the environmental conditions. A potato cultivar can induce resistance to a specific PVY strain, yet be susceptible to another. How a single potato cultivar responds to PVY in both compatible and incompatible interactions is not clear.

RESULTS

In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare the transcriptional changes in leaves of potato upon inoculation with PVY. We used two potato varieties: Premier Russet, which is resistant to the PVY strain O (PVY(O)) but susceptible to the strain NTN (PVY(NTN)), and Russet Burbank, which is susceptible to all PVY strains that have been tested. Leaves were inoculated with PVY(O) or PVY(NTN), and samples were collected 4 and 10 h post inoculation (hpi). A larger number of differentially expressed (DE) genes were found in the compatible reactions compared to the incompatible reaction. For all treatments, the majority of DE genes were down-regulated at 4 hpi and up-regulated at 10 hpi. Gene Ontology enrichment analysis showed enrichment of the biological process GO term "Photosynthesis, light harvesting" specifically in PVY(O)-inoculated Premier Russet leaves, while the GO term "nucleosome assembly" was largely overrepresented in PVY(NTN)-inoculated Premier Russet leaves and PVY(O)-inoculated Russet Burbank leaves but not in PVY(O)-inoculated Premier Russet leaves. Fewer genes were DE over 4-fold in the incompatible reaction compared to the compatible reactions. Amongst these, five genes were DE only in PVY(O)-inoculated Premier Russet leaves, and all five were down-regulated. These genes are predicted to encode for a putative ABC transporter, a MYC2 transcription factor, a VQ-motif containing protein, a non-specific lipid-transfer protein, and a xyloglucan endotransglucosylase-hydroxylase.

CONCLUSIONS

Our results show that the incompatible and compatible reactions in Premier Russet shared more similarities, in particular during the initial response, than the compatible reactions in the two different hosts. Our results identify potential key processes and genes that determine the fate of the reaction, compatible or incompatible, between PVY and its host.

Neuroinflammation is an essential mechanism involved in the pathogenesis of subarachnoid hemorrhage (SAH)-induced brain injury. Recently, Netrin-1 (NTN-1) is well established to exert anti-inflammatory property in non-nervous system diseases through inhibiting infiltration of neutrophil. The present study was designed to investigate the effects of NTN-1 on neuroinflammation, and the potential mechanism in a rat model of SAH. Two hundred and ninety-four male Sprague Dawley rats (weight 280-330 g) were subjected to the endovascular perforation model of SAH. Recombinant human NTN-1 (rh-NTN-1) was administered intravenously. Small interfering RNA (siRNA) of NTN-1 and UNC5B, and a selective PPARγ antagonist bisphenol A diglycidyl ether (BADGE) were applied. Post-SAH evaluations included neurobehavioral function, brain water content, Western blot analysis, and immunohistochemistry. Our results showed that endogenous NTN-1 and its receptor UNC5B level were increased after SAH. Administration of rh-NTN-1 reduced brain edema, ameliorated neurological impairments, and suppressed microglia activation after SAH, which were concomitant with PPARγ activation, inhibition of NFκB, and decrease in TNF-α, IL-6, and ICAM-1, as well as myeloperoxidase (MPO). Knockdown of endogenous NTN-1 increased expression of pro-inflammatory mediators and MPO, and aggravated neuroinflammation and brain edema. Moreover, knockdown of UNC5B using specific siRNA and inhibition of PPARγ with BADGE blocked the protective effects of rh-NTN-1. In conclusion, our findings indicated that exogenous rh-NTN-1 treatment attenuated neuroinflammation and neurological impairments through inhibiting microglia activation after SAH in rats, which is possibly mediated by UNC5B/PPARγ/NFκB signaling pathway. Exogenous NTN-1 may be a novel therapeutic agent to ameliorating early brain injury via its anti-inflammation effect.

Matrix metalloproteinase 9 (MMP9) is a conditionally expressed enzyme and is upregulated in glomerulonephritis. Its function in these diseases, however, remains to be fully elucidated. The induction of nephrotoxic serum nephritis (NTN) in wild-type mice resulted in an upregulation of MMP9, followed by leukocyte infiltration, albuminuria, and subsequent renal failure. MMP9 deficiency ameliorated the course of NTN as indicated by reduced histological injury and reduced infiltration of proinflammatory macrophages. The chemotaxis of MMP9-deficient macrophages in vitro was impaired. Intrarenal macrophages isolated from the kidneys of nephritic MMP9 knockout mice still displayed the typical features of a proinflammatory phenotype and were indistinguishable from wild type-derived cells. Bone marrow transplantation restored renal tissue injury and macrophage recruitment when wild type-derived donor cells were transplanted onto MMP9-deficient mice prior to the induction of NTN. Thus, leukocyte-derived MMP9 mediates the recruitment of proinflammatory macrophages into kidneys during experimental crescentic glomerulonephritis.

Hirschsprung's (HSCR) disease is a congenital intestinal malformation of the enteric nervous system. It is a multigenic malformation and until now, eight genes have been involved in the etiology of this disease: genes encoding proteins of the RET signaling pathway (RET, GDNF and NTN), genes participating in the endothelin (EDN) type B receptor pathway (EDNRB, EDN3 and ECE-1), the SOX10 gene and the SIP1 gene that is mutated in syndromic forms of HSCR. Mutations of these genes are found in not more than 50-60% of affected individuals. Here, we report on the results of a molecular cytogenetic study performed in a girl who presented with a syndromic short segment HSCR associated with a de novo t(4;8)(p13;p22) translocation. A comparative genomic hybridization (CGH) study found a 4p12p13 deletion. A molecular characterization of this rearrangement showed that the 4p13 deletion was 5 Mb in length and included the paired mesoderm homeobox gene (PMX2B) (MIM 603851), a gene expressed in the human embryonic gut and essential for the development of autonomic neural crest derivatives. The present observation suggests that PMX2B haploinsuffciency might predispose to HSCR.

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5-11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.

BACKGROUND

The L-aminopeptidase D-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal L and/or D-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both D and L stereospecificity. The DmpA active form is an alphabeta heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide.

RESULTS

The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an alphabetabetaalpha sandwich in which two mixed beta sheets are flanked on both sides by two alpha helices.

CONCLUSIONS

DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.

The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates.

Penicillium chrysogenum Acyl coenzyme A:isopenicillin N acyltransferase (AT) performs the last step in the biosynthesis of hydrophobic penicillins, exchanging the hydrophilic side chain of a precursor for various hydrophobic side chains. Like other N-terminal nucleophile hydrolases AT is produced as an inactive precursor that matures upon posttranslational cleavage. The structure of a Cys103Ala precursor mutant shows that maturation is autoproteolytic, initiated by Cys103 cleaving its preceding peptide bond. The crystal structure of the mature enzyme shows that after autoproteolysis residues 92-102 fold outwards, exposing a buried pocket. This pocket is structurally and chemically flexible and can accommodate substrates of different size and polarity. Modeling of a substrate-bound state indicates the residues important for catalysis. Comparison of the proposed autoproteolytic and substrate hydrolysis mechanisms shows that in both events the same catalytic residues are used, but that they perform different roles in catalysis.

The effect of neurotensin (NTN) on preventing microbial translocation and preserving intestinal mucosal integrity after abdominal radiation was studied in rats. Animals were divided into the following groups: I (control), II (radiation control) and III (radiation and NTN). Radiation (1,100 cGy) was administered on the 1st day to groups II and III. NTN (300 micrograms/kg) was given intraperitoneally to group III animals, once daily for 3 days. On the 4th day, mesenteric lymph nodes (MLN) were obtained and cultured. Villi per centimeter (V/cm), villus height (Vh) and mitoses per crypt (M/c) were evaluated from ileal mucosa. Radiation increased positive MLN cultures, while treatment with NTN reduced them significantly. V/cm and Vh also returned to normal levels after NTN treatment, while M/c were increased in all irradiated animals. It was shown that NTN reduces bacterial translocation after abdominal radiation. Examination of ileal mucosa indicates that this can be attributed to the improvement of the mucosal integrity, due to the trophic effect of the hormone on the gut.

Exaggerated pressor and muscle sympathetic nerve activity (MSNA) responses have been reported during static handgrip in hypertensive (HTN) adults. Recent work suggests that such responses may occur much more rapidly in HTN patients; however, this has not been extensively studied. Thus, we examined the blood pressure (BP) and MSNA responses at the immediate onset of muscle contraction and tested the hypothesis that older HTN adults would exhibit rapid onset pressor and sympathetic responses compared with normotensive (NTN) adults. Heart rate (HR), BP (Finometer) and MSNA (peroneal microneurography) were retrospectively analyzed in 15 HTN (62 ± 1 years; resting BP 153 ± 3/91 ± 5 mm Hg) and 23 age-matched NTN (60 ± 1 years; resting BP 112 ± 1/67 ± 2 mm Hg) subjects during the first 30 s of static handgrip at 30 and 40% of maximal voluntary contraction (MVC). HTN adults demonstrated exaggerated increases in mean BP during the first 10 s of both 30% (NTN: Δ1 ± 1 vs HTN: Δ7 ± 2 mm Hg; P < 0.05) and 40% (NTN: Δ2 ± 1 vs HTN: Δ8 ± 2 mm Hg; P < 0.05) intensity handgrip. Likewise, HTN adults exhibited atypical increases in MSNA within 10 s. Increases in HR were also greater in HTN adults at 10 s of 30% MVC handgrip, although not at 40% MVC. There were no group differences in 10 s pressor or sympathetic responses to a cold pressor test, suggesting no differences in generalized sympathetic responsiveness. Thus, static handgrip evokes rapid onset pressor and sympathetic responses in older HTN adults. These findings suggest that older HTN adults likely have greater cardiovascular risk even during short duration activities of daily living that contain an isometric component.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.

Zebrafish no tectal neuron (ntn) mutant obtained by trimethylpsoralen (TMP) mutagenesis showed defects in tectal neuropil formation and small eyes. We carried out whole-genome subtraction between wild-type and mutant zebrafish embryos using the representational difference analysis (RDA) method. Nineteen subtraction products enabled us to construct genetic and physical maps of the ntn region. Direct selection of cDNAs using a YAC clone encompassing the ntn locus and RT-PCR analysis of transcripts identified a 143 bp deletion in the cct3 gene encoding the gamma subunit of chaperonin containing TCP-1 (CCT). Injection of antisense cct3 morpholino oligonucleotides into zebrafish embryos induced characteristic ntn phenotypes including defects in retinal ganglion cell (RGC) differentiation and tectal neuropil formation. Moreover, injection of cct3 mRNA successfully rescued ntn mutant embryos. Our results suggest that RDA is an efficient and widely applicable cloning strategy in zebrafish genetics. The strong expression of the cct3 mRNA started in the entire embryos by 12 hpf and was sustained thereafter, but there were no detectable abnormalities in body patterning and neurogenesis in ntn mutant embryos at 30 hpf. The expression patterns of transcription factor genes ath5 and brn3b that are essential for the development and maintenance of RGCs were indistinguishable between wild-type and ntn mutant embryos, but those of early and late differentiation markers of RGCs, nicotinic acetylcholine receptor beta 3 and zn5, were diminished in mutant embryos. Immunostaining of acetylated tubulin also revealed the impairment of RGC neurite extension. Thus, the ntn mutation of the cct3 gene impaired the differentiation of retinal neuroepithelial cells to RGCs. Similarly, the expression of brn3b was normal in the tectum of ntn mutants, but tectal neuropil formation was abolished. These results suggest that the gamma subunit of chaperonin CCT plays an essential role in retinotectal development.

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.

Glomerular antibody deposition induces acute neutrophil-mediated glomerular injury via activation of c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). However, the link between antibody deposition and activation of JNK/p38 MAPK signalling is unclear. This study tested the postulate that spleen tyrosine kinase (Syk), which is activated via Fcγ-receptor ligation, is required for activation of JNK and p38 signalling and acute neutrophil-mediated glomerular injury. We used a Syk inhibitor (SYKi) in rat nephrotoxic serum nephritis (NTN) in which neutrophil-mediated glomerular injury is dependent upon JNK and p38 signalling. SYKi or vehicle treatment of Sprague-Dawley rats began 30 min before administration of anti-GBM serum with rats killed 3 or 24 h later. Immunostaining identified de novo glomerular Syk activation (p-Tyr 525/526) in untreated NTN, being most prominent in neutrophils. Vehicle and untreated NTN exhibited heavy proteinuria and glomerular thrombosis at 24 h with P-selectin and fibrin immunostaining within capillaries, glomerular macrophage and T cell infiltration, activation of JNK and p38 MAPK signalling, and upregulation of glomerular mRNA levels of pro-inflammatory molecules (TNF-α, NOS2, MMP-12 and CCL2). In contrast, SYKi treatment provided complete protection from proteinuria, with a profound reduction in glomerular thrombosis and immunostaining for P-selectin and fibrin, and a substantial reduction in glomerular mRNA levels of pro-inflammatory molecules. SYKi treatment also reduced the acute glomerular neutrophil influx and pro-inflammatory response at 3 h in NTN. These protective effects were associated with a significant reduction in glomerular JNK and p38 MAPK activation. In addition, activation of Syk, JNK and p38 was identified in human biopsy samples of acute crescentic glomerulonephritis. In conclusion, this study demonstrates that Syk signalling is required for JNK and p38 MAPK signalling and acute neutrophil-dependent glomerular injury in rat NTN. These findings identify Syk as a potential therapeutic target in antibody-dependent kidney disease.