The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The approximately 17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The approximately 1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located approximately 500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.

BACKGROUND

N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712.

RESULTS

The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases.

CONCLUSIONS

The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like β-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and β-subunits, with the catalytic serine as the first amino acid of the β-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.

Hirschsprung's disease is an inherited disorder showing incomplete penetrance and variable expressivity. Genetic mapping and mutation screening of candidate genes, together with the study of several natural and knockout animal models, clearly have shown the involvement of several different genes in the pathogenesis of Hirschsprung's disease. Among these genes, the RET proto-oncogene accounts for the highest proportion of both familial and sporadic cases, with a wide range of mutations scattered along its entire coding region. The low detection rate of RET mutations in Hirschsprung patients also led to different hypotheses, such as the existence of additional Hirschsprung genes. Different animal and human genetic studies have identified 6 Hirschsprung genes: RET proto-oncogene (RET), endothelin 3 (EDN3), endothelin B receptor gene (EDNRB), glial-cell-line-derived neurotrophic factor (GDNF), endothelin converting enzyme (ECE1), gene encoding the Sry-related transcription factor SOX10 (SOX10). Microenvironmental factors also can play a role in the pathogenesis of aganglionosis. The developmental process of the crest-derived progenitor cells is sensitive to the level of different molecules. The expression deficit of different factors (GDNF, NTN) in the hindgut, in the absence of genetic mutations, could determine a missed activation of the receptor system, causing enteric neuroblast migration arrest.

OBJECTIVE

Parkinson's disease is characterized by profound motor deficits that result mainly as a consequence of degeneration of midbrain dopaminergic neurons. No current therapy slows or halts disease progression. Neurturin (NTN) and glial cell line-derived neurotrophic factor have potent neuroprotective and neurorestorative effects on dopaminergic neurons, but their use in treating Parkinson's disease has been limited by significant delivery obstacles. In this study, we examined the long-term expression, bioactivity, and safety/tolerability of CERE-120, an adeno-associated virus type 2 vector encoding human NTN, after bilateral stereotactic delivery to the striatum of nonhuman primates.

METHODS

Twelve naïve rhesus macaques received bilateral stereotactic injections of 1 of 2 CERE-120 doses or vehicle to the caudate and putamen. Neurological and clinical parameters were monitored for up to 1 year postadministration, after which animals were sacrificed for histological analyses.

RESULTS

Dose-related NTN expression was observed at 1 year and was associated with enhanced tyrosine hydroxylase immunolabeling in the striatum, hypertrophy of tyrosine hydroxylase-positive cells in the substantia nigra, and induction of extracellular signal-regulated kinase signaling in the substantia nigra. Extensive, formal analyses, conducted in accordance with Good Laboratory Practice Regulations, across multiple time points revealed no evidence of clinical, neurological, or systemic toxicity.

CONCLUSIONS

The present study provides evidence of long-term expression and bioactivity of NTN on the dopaminergic nigrostriatal system after bilateral stereotactic delivery of CERE-120 to the striatum. Furthermore, no evidence of any adverse effects for up to 1 year postadministration was observed. These findings reveal a wide safety margin for CERE-120 and collectively support the ongoing clinical testing of the efficacy and safety of CERE-120 in patients with Parkinson's disease.

OBJECTIVE

In the early stages of hypertension (HTN), when global left ventricular (LV) function is still unaffected, localized geometrical changes suggest changes in regional function. We investigated regional geometry and systolic deformation (using strain/strain rate (S/SR) imaging) in HTN.

RESULTS

We studied 74 untreated mild to moderate HTNs and 34 matched normotensives (NTN). All had a standard echo including myocardial velocity data for regional radial and longitudinal deformation. Despite the absence of abnormalities in standard functional indices and LVH, non-uniform changes in regional geometry and deformation were observed. Besides a significant increase in wall thickness (WT) in all HTN segments, there was a gradual increase in WT from apex to base resulting in prominent basal septal hypertrophy. In HTN, regional longitudinal peak systolic SR (SSR) and end-systolic S (ESS) were significantly (P < 0.0001) reduced in the basal septum. In the lateral wall there was an increase in peak SSR and ESS (P < 0.05) basally. The basal septal ESS correlated both with mean arterial pressure and basal septal WT, with lower ESS for higher BP and thicker septum.

CONCLUSIONS

Regionally differing geometrical remodelling occurs early in HTN. Longitudinal ESS and peak SSR are sensitive markers of early changes occurring in HTN.

In many Gram-negative pathogens, their virulent behavior is regulated by quorum sensing, in which diffusible signals such as N-acyl homoserine lactones (AHLs) act as chemical messaging compounds. Enzymatic degradation of these diffusible signals by, e.g., lactonases or amidohydrolases abolishes AHL regulated virulence, a process known as quorum quenching. Here we report the first crystal structure of an AHL amidohydrolase, the AHL acylase PvdQ from Pseudomonas aeruginosa. PvdQ has a typical alpha/beta heterodimeric Ntn-hydrolase fold, similar to penicillin G acylase and cephalosporin acylase. However, it has a distinct, unusually large, hydrophobic binding pocket, ideally suited to recognize C12 fatty acid-like chains of AHLs. Binding of a C12 fatty acid or a 3-oxo-C12 fatty acid induces subtle conformational changes to accommodate the aliphatic chain. Furthermore, the structure of a covalent ester intermediate identifies Serbeta1 as the nucleophile and Asnbeta269 and Valbeta70 as the oxyanion hole residues in the AHL degradation process. Our structures show the versatility of the Ntn-hydrolase scaffold and can serve as a structural paradigm for Ntn-hydrolases with similar substrate preference. Finally, the quorum-quenching capabilities of PvdQ may be utilized to suppress the quorum-sensing machinery of pathogens.

OBJECTIVE

To examine the ability of injection of C-reactive protein (CRP) to treat systemic lupus erythematosus (SLE) in the (NZB x NZW)F(1) (NZB/NZW) mouse and to use a nephrotoxic nephritis (NTN) model to further examine the mechanism of this activity.

METHODS

NZB/NZW mice were given a single injection of 200 mug of CRP prior to disease onset or after the onset of high-grade proteinuria. Mice were monitored weekly for proteinuria and monthly for anti-double-stranded DNA (anti-dsDNA) antibodies. NTN was induced by immunization with rabbit IgG followed by rabbit anti-mouse glomerular basement membrane. Proteinuria was measured daily, and renal pathology was scored. CRP was injected at the time of disease induction or 9 days later.

RESULTS

Treatment of NZB/NZW mice with CRP prior to disease onset delayed the onset of high-grade proteinuria by 16 weeks (P < 0.0001) and prolonged survival by 13 weeks (P < 0.002). CRP treatment of NZB/NZW mice during acute disease rapidly decreased proteinuria, and the treated mice remained aproteinuric for at least 10 weeks. Control and CRP-treated mice developed similar levels of anti-dsDNA. In C57BL/6 mice, injection of CRP either before or after induction of NTN suppressed proteinuria and glomerular pathology. CRP was completely ineffective in treating NTN in interleukin-10 (IL-10)-deficient mice.

CONCLUSIONS

CRP injection suppresses inflammation in the kidney in SLE and NTN. The requirement for IL-10 in this protection suggests that CRP must rapidly initiate an IL-10-dependent antiinflammatory process. These findings suggest that a major function of CRP during the acute-phase response is to limit tissue damage and modulate acute inflammation.

The role of polymorphonuclear leucocytes (PMN) in the autologous phase of nephrotoxic nephritis (NTN) in the rabbit has been investigated. Depletion of circulating PMN by nitrogen mustard protected renal function and immunofluorescent examination showed reduction in glomerular fibrin deposition. Depletion of circulating PMN using a highly specific goat anti-PMN serum (APS) provided similar protection of renal function, highly significant reduction in proteinuria and histological and immunofluorescent examination showed reduced glomerular PMN infiltration, extracapillary cell proliferation and virtual absence of fibrin deposition. Although protection by nitrogen mustard may have been partly due to immunosuppression, no reduction in antibody response was detected in the APS-treated rabbits. The results implicate the polymorph as the principal injurious agent in this model of NTN, responsible directly or indirectly for both proteinuria and glomerular fibrin deposition.

hUC-MSCs hold great promise in vitro neuronal differentiation and therapy for neurodegenerative disorders including Parkinson's disease. Recent studies provided that Lmx1α play an important role in the midbrain dopamine cells differentiation. Neurturin is desired candidate gene for providing a neuroprotective to DA neurons. In this study, we investigated a novel neuronal differentiation strategy in vitro with Lmx1α and NTN. We transferred these two genes to hUC-MSCs by recombinant adenovirus combined with Lmx1α regulatory factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored by using behavioral test for six months after implantation. The result showed that cells isolated from the umbilical cord were negative for CD45, CD34 and HLA-DR, but were positive for CD44, CD49d, CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western blot. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. After hUC-MSCs were induced with endogenous and exogenous factors, the mature neurons specific gene TH, Pitx3 was transcripted and the neurons specific protein TH, β-tubulinIII, NSE, Nestin, MAP-2 was expressed in those differentiated cells. In addition, the PD monkeys, transplanted with the induced cells demonstrated the animals' symptoms amelioration by the behavioral measures. Further more, pathological and immunohistochemistry data showed that there were neuronal-like cells survived in the right brain of those PD monkeys, which may play a role as dopaminergic neurons. The findings from this study may help us to better understand the inside mechanisms of PD pathogenesis and may also help developing effective therapy for Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.

The exercise pressor reflex (EPR) is composed of the mechanoreflex and the metaboreflex and has been shown to be overactive in spontaneously hypertensive rats. The aim of the present study was to isolate the metaboreflex using post-exercise ischemia (PEI) and examine the BP response in normotensive (NTN) and hypertensive (HTN) humans. We hypothesize that the post-exercise ischemia-induced maintenance of BP will be greater in HTN when compared to NTN adults. A total of 15 NTN (65 +/- 1 years) and 12 HTN (64 +/- 1 years) adults were recruited. Beat-to-beat mean arterial pressure (MAP) was measured non-invasively (Finometer). Dynamic handgrip exercise (DHE) was performed for 3 min followed by 2 min of PEI. An unpaired t test was used to examine differences between groups. As compared to resting baseline values, the change in MAP during PEI was greater in HTN than NTN subjects (HTN: Delta = 12 +/- 3 mmHg, NTN: Delta = 6 +/- 1 mmHg, P < 0.05). These data suggest that HTN humans have enhanced metaboreflex sensitivity.

The blood-brain barrier (BBB) constitutes a major obstacle in brain drug delivery. Focused ultrasound (FUS) in conjunction with microbubbles has been shown to open the BBB noninvasively, locally, and transiently to allow large molecules diffusion. Neurturin (NTN), a member of the glial-derived neurotrophic factor (GDNF) family, has been demonstrated to have neuroprotective and regenerative effects on dopaminergic neurons in vivo using invasive drug delivery methods. The brain's ascending nigrostriatal pathway is severely damaged in Parkinson's disease (PD), and therefore the substantia nigra (SN) and striatal caudoputamen (CP) were selected as the target areas. The objective of the study was to investigate whether safe and efficient NTN delivery can be achieved through FUS-induced BBB opening via intravenous administration, and thus trigger the neuroregeneration cascade in the nigrostriatal pathway. After the optimization of FUS parameters and target locations in the murine brain, NTN bioavailability and downstream signaling were detected and characterized through immunostaining. FUS significantly enhanced the delivery of NTN compared with the direct injection technique, whereas triggering of the signaling cascade was detected downstream to the neuronal nuclei. These findings thus indicate the potential of the FUS method to mediate transport of proteins through the blood-brain barrier in a PD animal model.

Heat shock proteins (Hsp) are ubiquitous intracellular proteins that can be released in various forms of cellular stress. Some Hsp, such as Hsp60, have been shown to stimulate directly T cell-mediated immune responses in vitro. Here, it is demonstrated that Hsp60 is released from the kidneys and excreted into the urine of mice with nephrotoxic nephritis (NTN), a model of rapidly progressive glomerulonephritis. For examining the functional relevance of Hsp60 release, this protein was injected into mice with subnephritogenic NTN, in which only transient proteinuria and minimal organ damage occur that do not progress to terminal kidney failure. Injection of Hsp60 strikingly aggravated disease, as evidenced by global glomerular necrosis, tubulointerstitial damage, and complete anuria after 10 to 12 d. This effect was mediated neither by endotoxin contaminations of Hsp60 nor by autologous antibodies. It was strictly T cell dependent but not associated with a systemic Th1/Th2 shift. Thus, Hsp60 is an endogenous mediator stimulating immune effector mechanisms that contribute to the progression of NTN. These findings demonstrate in vivo that Hsp60 fulfills criteria of immunologic danger signals and suggest that such signals may be involved in immune-mediated kidney disease.

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

The role played by the macrophage in the development of injury in rabbit nephrotoxic nephritis (NTN) has been assessed by electron microscopy and glomerular culture of renal tissue obtained by several biopsies during the course of the disease. These observations have been correlated with the other immune, cellular, and biochemical events occurring in the glomerulus, ie, deposition of immunoglobulin and complement, proteinuria, polymorphonuclear leukocyte (PMN) exudation, fibrin deposition, crescent formation, and renal failure. A biphasic macrophage accumulation was detected, corresponding to the heterologous and autologous phases of the disease. In the autologous or crescentic phase, macrophages accumulated within the glomerular tuft from Day 5; their appearance coincided with the accumulation of PMN and development of proteinuria. Fibrin deposition in Bowman's space, which commenced on Days 6 and 7, was rapidly followed by the migration of macrophages from the glomeruli into Bowman's space. Within Bowman's space, macrophages were observed to phagocytose fibrin, transform into epithelioid and giant cells, and accumulate to form a substantial proportion of the cells forming the crescent. The inflammatory process of PMN exudation, macrophage accumulation, fibrin deposition, and crescent formation and the degree of renal failure reached a maximum by Days 12 to 14. Thereafter, resolution of the inflammatory process occurred so that by Day 40 macrophages had disappeared from the glomeruli. However, varying degrees of glomerular damage and renal failure persisted, occurring largely as a result of glomerulosclerosis and sclerosis of crescents. The tissue culture studies also demonstrated mesangial cell proliferation during the inflammatory process but did not show any abnormality of epithelial cell activity. This study demonstrates that the macrophages participate in NTN by accumulating in damaged glomeruli then migrating into Bowman's space (probably in response to fibrin deposition) where they undergo granulomatous transformation and accumulate, contributing to crescent formation.

The proteasome is the central enzyme of protein degradation in the cytosol and the nucleus. It is involved in the removal of abnormal, misfolded or incorrectly assembled proteins, in the processing or degradation of transcriptional regulators in stress response, in degradation of cyclins in cell-cycle control, in the destruction of transcription factors or metabolic enzymes in cell differentiation and metabolic response, and in MHC class I mediated cellular immune response. By the analysis of the crystal and molecular structures of the 20 S proteasomes from the archaeon Thermoplasma acidophilum and from yeast it was shown that the beta-type subunits in which the proteolytic activities reside are members of the N-terminal nucleophile (Ntn) protein family. They are synthesized as proproteins and become active by autoprocessing at a Gly-1-Thr1 bond. The Thr1Ala mutant of subunit beta1/Pre3 of the 20 S proteasome from yeast is unable to autolyse. Its crystal and molecular structure at 2.2 A resolution described here shows that the pro-segment adopts a well-defined gamma-turn conformation at Gly-1 and provides a first view at an autolysis site in Ntn hydrolases. The Gly-1 carbonyl oxygen displays two hydrogen bonds. The modelled Thr1 side-chain is located above the gamma-turn bulge such that addition of its nucleophilic hydroxyl group to the electrophilic Gly-1 carbonyl carbon atom may proceed by very small motions. The pro-segment binding site and the catalytic site provide a rigid structural framework and appropriate hydrogen bond donors for this reaction. The same structure also supports addition of the Thr1 hydroxyl group to the carbonyl carbon atom of Leu-2 as a model for the first step in substrate hydrolysis by the proteasome.

Macrophages are intimately involved in the development of immune-mediated inflammation, including glomerulonephritis. We have transduced primary cultures of macrophages to express IL-10 and tested the ability of these cells to control rat nephrotoxic nephritis (NTN), a model of human glomerulonephritis. Ad-IL-10-transduced bone-marrow-derived macrophages (BMDM) produced large amounts of IL-10 in culture, and their TNF-alpha production was decreased in response to interferon-gamma and LPS. Transduced macrophages were injected into the renal artery of rats, 6 h after the induction of NTN, where they localized efficiently to inflamed rat glomeruli. Delivery of IL-10-expressing macrophages to nephritic rats produced a marked reduction in albuminuria compared with unmodified NTN or injection of Ad-null-transduced BMDM. IL-10 treatment decreased the number of glomerular ED1- and ED3-positive cells, MHC class II expression, and the number of fibrinoid lesions. Interestingly, anti-inflammatory changes in the Ad-IL-10-injected kidney were mirrored by changes in the contralateral kidney. These results highlight that Ad-IL-10-transduced macrophages infiltrate inflamed glomeruli and reduce the severity of glomerular inflammation, emphasizing the value of local delivery of genetically modified macrophages in the manipulation of inflammatory disease.

A pathogenic role for Th17 cells in inflammatory renal disease is well established. The mechanisms underlying their counter-regulation are, however, largely unknown. Recently, Th17 lineage-specific regulatory T cells (Treg17) that depend on activation of the transcription factor Stat3 were identified. We studied the function of Treg17 in the nephrotoxic nephritis (NTN) model of crescentic GN. The absence of Treg17 cells in Foxp3(Cre)×Stat3(fl/fl) mice resulted in the aggravation of NTN and skewing of renal and systemic immune responses toward Th17. Detailed analysis of Stat3-deficient Tregs revealed that the survival, activation, proliferation, and suppressive function of these cells remained intact. However, Tregs from Foxp3(Cre)×Stat3(fl/fl) mice lacked surface expression of the chemokine receptor CCR6, which resulted in impaired renal trafficking. Furthermore, aggravation of NTN was reversible in the absence of Th17 responses, as shown in CD4(Cre)×Stat3(fl/fl) mice lacking both Treg17 and Th17 cells, suggesting that Th17 cells are indeed the major target of Treg17 cells. Notably, immunohistochemistry revealed CCR6-bearing Treg17 cells in kidney biopsy specimens of patients with GN. CCR6 expression on human Treg17 cells also appears dependent on STAT3, as shown by analysis of Tregs from patients with dominant-negative STAT3 mutations. Our data indicate the presence and involvement of Stat3/STAT3-dependent Treg17 cells that specifically target Th17 cells in murine and human crescentic GN, and suggest the kidney-specific action of these Treg17 cells is regulated by CCR6-directed migration into areas of Th17 inflammation.

Host gene expression changes in the early response to potato virus Y(NTN) interaction were compared in two differently sensitive potato cultivars: the resistant cultivar Santé and the sensitive cultivar Igor. Hybridization of potato TIGR cDNA microarrays allowed us to monitor the expression of approximately 10,000 genes simultaneously at 0.5 and 12 h post-inoculation (hpi). Microarray data, analysed by statistics and data mining, were complemented by subtraction library construction and sequence analysis to validate the findings. The expression profiles of the two cultivars were similar and faint at 0.5 hpi, but they differed substantially at 12 hpi. Although, at 0.5 hpi, cv. Santé responded by the differential expression of a greater number of genes, at 12 hpi the number was higher in cv. Igor. The majority of genes in this cultivar were down-regulated at 12 hpi, indicating a host gene shut-off. Suites of genes that exhibited altered transcript abundance in response to the virus were identified, and included genes involved in the processes of photosynthesis, perception, signalling and defence responses. The expression of the considerable number of genes associated with photosynthesis was surprisingly up-regulated as early as 0.5 hpi and down-regulated at 12 hpi in both cultivars. The expression of genes involved in perception and signalling was increased in the sensitive cultivar at 12 hpi. By contrast, a simultaneous strong defence response at the transcriptional level was evident in the resistant cultivar, as shown by the up-regulation of genes involved in brassinosteroid, polyamine and secondary metabolite biosynthesis, and of genes coding for pathogenesis-related proteins.

Establishment of the neuromuscular synapse requires bidirectional signaling between the nerve and muscle. Although much is known on nerve-released signals onto the muscle, less is known of signals important for presynaptic maturation of the nerve terminal. Our results suggest that the Ret tyrosine kinase receptor transmits a signal in motor neuron synapses that contribute to motor neuron survival and synapse maturation at postnatal stages. Ret is localized specifically to the presynaptic membrane with its ligands, GDNF (glial cell line-derived neurotrophic factor)/NTN (neurturin), expressed in skeletal muscle tissue. Lack of Ret conditionally in cranial motor neurons results in a developmental deficit of maturation and specialization of presynaptic neuromuscular terminals. Regeneration of Ret-deficient adult hypoglossal motor neurons is unperturbed, but despite contact with the unaffected postsynaptic specializations, presynaptic axon terminal maturation is severely compromised in the absence of Ret signaling. Thus, Ret transmits a signal in motor nerve terminals that participate in the organization and maturation of presynaptic specializations during development and during regeneration in the adult.

Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [(3)H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6β4, except uncoordinated family member (UNC) 5A-C, and neogenin. Among them, IN α6β4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3β (GSK-3β) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3β were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and formation of cyt c and HSP27 complex. Moreover, the inhibition of each signaling protein attenuated Ntn-1-induced blockage of apoptosis. In conclusion, Ntn-1-induced HSP27 protected hypoxic injury-related UCB-MSC apoptosis through DCC- and IN α6β4-dependent Akt, GSK-3β, and HSF-1 signaling pathways.

Bone morphogenic protein (BMP)-7 is expressed in the adult kidney and reverses chronic renal injury when given exogenously. Here, we report that a histone deacetylase inhibitor, trichostatin A (TSA), attenuates chronic renal injury, in part, by augmenting the expression of BMP-7 in kidney side population (SP) cells. We induced accelerated nephrotoxic serum nephritis (NTN) in C57BL/6 mice and treated them with TSA for 3 weeks. Compared with vehicle-treated NTN mice, treatment with TSA prevented the progression of proteinuria, glomerulosclerosis, interstitial fibrosis, and loss of kidney SP cells. Basal gene expression of renoprotective factors such as BMP-7, vascular endothelial growth factor, and hepatocyte growth factor was significantly higher in kidney SP cells as compared with non-SP cells. Treatment with TSA significantly upregulated the expression of BMP-7 in SP cells but not in non-SP cells. Moreover, initiation of treatment with TSA after 3 weeks of NTN (for 3 weeks, until 6 weeks) partially but significantly reversed renal dysfunction. Our results indicate an important role of SP cells in the kidney as one of the possible generator cells of BMP-7 and TSA as a stimulator of the cells in reversing chronic renal disease. Disclosure of potential conflicts of interest is found at the end of this article.