Environmental impacts of nitrogen emissions in China and the role of policies in emission reduction.

Atmospheric reactive nitrogen (Nr) has been a cause of serious environmental pollution in China. Historically, China used too little Nr in its agriculture to feed its population. However, with the rapid increase in N fertilizer use for food production and fossil fuel consumption for energy supply over the last four decades, increasing gaseous Nr species (e.g. NH3 and NOx) have been emitted to the atmosphere and then deposited as wet and dry deposition, with adverse impacts on air, water and soil quality as well as plant biodiversity and human health. This paper reviews the issues associated with this in a holistic way. The emissions, deposition, impacts, actions and regulations for the mitigation of atmospheric Nr are discussed systematically. Both NH3 and NOx make major contributions to environmental pollution but especially to the formation of secondary fine particulate matter (PM2.5), which impacts human health and light scattering (haze). In addition, atmospheric deposition of NH3 and NOx causes adverse impacts on terrestrial and aquatic ecosystems due to acidification and eutrophication. Regulations and practices introduced by China that meet the urgent need to reduce Nr emissions are explained and resulting effects on emissions are discussed. Recommendations for improving future N management for achieving 'win-win' outcomes for Chinese agricultural production and food supply, and human and environmental health, are described. This article is part of a discussion meeting issue 'Air quality, past present and future'.

A novel mutation links to von Hippel-Lindau syndrome in a Chinese family with hemangioblastoma.

Hemangioblastoma of the central nervous system occurs as sporadic tumors or as a part of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary tumor syndrome caused by a germline mutation in the VHL tumor suppressor gene. We screened a Chinese family with VHL for mutations in the VHL gene and evaluated a genetic test for diagnosing VHL disease and clinical screening of family members. DNA extracted from the peripheral blood of all live members and from tissue of deceased family members with VHL disease was amplified by polymerase chain reaction to 3 VHL gene exons. Mutations in the amplification products were compared against the Human Gene Mutation Database. The involvement of multiple organs among the kindred with VHL disease was confirmed by medical history and radiography. Of the 12 members of the 4-generation family, 5 were diagnosed with VHL disease. Patient age at the initial diagnosis was 26-36 years (mean = 31 years). The mean time was 15 (11-19 months) from symptom appearance to the first patient visit to the hospital. Sequence analysis revealed that the frameshift mutation 327del C (p.Gly39Alafs*26) in exon 1 affected all family members, but not the healthy individuals or 16 unrelated controls. Members without gene mutation showed no clinical manifestation of VHL disease. We detected a conserved novel frameshift mutation in the VHL gene of the family members that contributes to VHL. DNA analysis of VHL is advantageous for VHL diagnosis. We developed a quick and reliable method for VHL diagnosis.

Shear stress modulates endothelial KLF2 through activation of P2X4.

Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.

The human P2X7 receptor and its role in innate immunity.

The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel/pore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.

Sexual function and health-related quality of life following anterior vaginal wall surgery for stress urinary incontinence and pelvic organ prolapse.

To assess female sexual function (FSF) and health-related quality of life (HRQOL) following anterior vaginal wall surgeries for stress urinary incontinence (SUI) and pelvic organ prolapse (POP). The retrospective study consisted of 116 patients. Chinese translations of the modified Lemack Questionnaire (not validated) and Pelvic Floor Distress Inventory-Short Form 20 were used to assess FSF and HRQOL, 3 months pre-operatively and 12-24 months (mean 16.8 months) post-operatively. Sixty-one (52.6%, 29 in SUI group and 32 in POP group) of patients were sexually active before and after the operation. Overall, 12 (19.7%, six in SUI group and six in POP group) reported an improvement in overall intercourse satisfaction, 21 (34.4%, 8 in the SUI and 13 in the POP group) were decreased and 28 (45.9%, 15 in SUI group and 13 in POP group) were unchanged. Incidence of coital incontinence decreased significantly in SUI group. Frequency of intercourse decreased, vaginal dryness and pain due to it and asymptomatic vaginal narrowing increased significantly, following the surgery in POP group. There were no statistically significant differences in the frequency of intercourse in SUI group, patients' perception of intercourse, frequency of orgasm and the importance of sex life in both groups. Partner discomfort remained unchanged. HRQOL improved significantly after the operation in both groups. There was no association between HRQOL and FSF in the post-operative period. In most patients, overall FSF did not impaired. All trans-anterior vaginal wall surgery positively impacted on the patients' HRQOL. A prospective study with validated questionnaire is necessary in future.

Urethral cystoscopic realignment and early end-to-end anastomosis develop different influence on erectile function in patients with ruptured bulbous urethra.

The objective of this study is to compare the influence on erectile function between urethral cystoscopic realignment and early end-to-end anastomosis treating ruptured bulbous urethra. 58 cases were selected, 32 had urethral cystoscopic realignment (group I) and 26 cases had urethral end-to-end anastomosis (group II). The parameters of P-CDU (Penile Color Duplex Ultrasound), NPT (Nocturnal Penile Tumescence), and IIEF-5 (International Index of Erectile Function) were compared between the two groups 6 months after operation. Group I was higher than group II in IIEF (21 vs 14) with significant differences. With P-CDU we observed an improvement in penile vascularization in group I as confirmed by the detection of an increase in peak systolic velocity (PSV) (26 cm/s vs 16 cm/s, p<0.01) and a decrease in end diastolic velocity (EDV) (3 cm/s vs 6 cm/s p<0.05), resulting in an increase in resistive index (RI) (0.85 vs 0.56, p<0.05). The parameters of NPT showed a significant increase compared to group II (p<0.01) in satisfactory erection number (5 vs 1.5), maximum rigidity (80% vs 42%), and total time that the increase in circumference was greater than 30% of baseline during sleep (100 sec vs 30 sec). Urethral cystoscopic realignment treating ruptured bulbous urethra can reduce the incidence of erectile dysfunction [ED]. A long term follow-up should be studied.

Reduced semen quality in chronic prostatitis patients that induce the release of apoptotic protein Omi/HtrA2 from spermatozoa.

The relationship between chronic prostatitis and fertility has been disputed for many years. Several groups have shown infection and autoimmune response against prostate antigens could have a deleterious effect on semen quality and fertility. This study was conducted to test the hypothesis that Omi/HtrA2-induced apoptosis in chronic prostatitis could be a mechanism underlying the observed clinical benefit. The Omi/HtrA2 serine protease is a nuclear-encoded mitochondrial protein, which can be released from mitochondria into the cytosol after apoptosis stimuli, inducing apoptosis in caspase-dependent and independent manners. Forty-one patients diagnosed as suffering from chronic prostatitis were included. Healthy normal individuals were included as controls. Human spermatozoa in the semen were purified by Percoll-gradient technique to separate the seminal plasma and other round cells. Measurements for sperm concentration, motility, morphology, proinflammatory cytokines, Omi/HtrA2 mRNA and protein levels in spermatozoa of chronic protatitis patients, were performed accordingly. Significantly increased levels of proinflammatory cytokines were detected in seminal plasma from these prostatitis patients. Omi/HtrA2 mRNA and protein levels were significantly higher in prostatitis men than in normal men. This study shows that chronic prostatitis patients present important alterations in their semen quality parameters, Omi/HtrA2 mRNA and protein levels of spermatozoa. We speculate that the inflammatory process involved may affect male fertility by release of proapoptotic protein Omi/HtrA2.

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes.

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.

Effect of apomorphine on intracavernous pressure and blood pressure in conscious, spinalized rats.

Apomorphine, given subcutaneously (s.c.), induces erection and bladder overactivity in rats through stimulation of dopamine (D1- and D2-like) receptors in the central nervous system. In paraplegic patients, apomorphine was reported to cause bladder overactivity. This suggests that apomorphine may have a spinal site of action also for stimulation of erection. The present study was initiated to evaluate the effect of apomorphine on erectile function in spinalized rats. Apomorphine (100 microg/kg, s.c.) was given to awake, unrestrained male Sprague-Dawley rats (300 g) with or without spinal cord injury, made at the Th 8 level 2 weeks before the experiment. Intracavernous pressure changes from baseline were evaluated as time to first response to apomorphine (TFR; sec), number of phasic pressure changes in the first 30 min (PP30), duration (D; sec) of the phasic pressure changes, the amount of increase in tonic peak pressure (TPP; cmH2O), and burst peak pressure (BPP; cmH2O). Blood pressure (cmH2O) was recorded via an intra-arterial catheter. Apomorphine, 100 microg/kg, caused no significant differences in TFR (217.8 vs 271.2), PP30 (6.4 vs 6.5), D (38.9 vs 37.6.), TPP (51.0 vs 54.0) and BPP (128.9 vs 160.4) between normal (n=8) and spinalized rats (n=6). However, blood pressure decreased significantly more in spinalized than in normal animals (17.7 vs 43.3; P<0.05). The results suggest that both in normal rats, and in rats with spinal cord injury, apomorphine given s.c., can produce erection. This finding supports the use of apomorphine for treatment of erectile dysfunction in paraplegia patients. However, due consideration should be given to possible decreases in blood pressure.

Point mutations confer loss of ATP-induced human P2X(7) receptor function.

Residues considered essential for ATP binding to the human P2X(7) receptor (hP2X(7)R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or site-directed mutants of hP2X(7)R constructs and channel/pore activity measured in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X(7)R.

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.

Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X(7) receptor (previously termed P2Z). These responses include opening of a cation-selective channel/pore that allows entry of the fluorescent dye ethidium and activation of a membrane metalloprotease that sheds the adhesion molecule L-selectin. The surface expression of P2X(7) receptors was measured in normal leucocytes, platelets, and B-CLL lymphocytes and correlated with their functional responses. Monocytes showed four- to fivefold greater expression of P2X(7) than B, T, and NK lymphocytes, whereas P2X(7) expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed lymphocyte P2X(7) at about the same level as B lymphocytes from normal subjects. P2X(7) function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes (n = 47, r = 0.70; P < 0.0001). However, in three patients the ATP-induced uptake of ethidium into the malignant B lymphocytes was low or absent. The lack of P2X(7) function in these B lymphocytes was confirmed by the failure of ATP to induce Ba(2+) uptake into their lymphocytes. This lack of function of the P2X(7) receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule that directs the recirculation of lymphocytes from blood into the lymph node.

Role of supraspinal tachykinins for micturition in conscious rats with and without bladder outlet obstruction.

In order to clarify the role of supraspinal tachykinins in volume-induced micturition and in bladder hyperactivity secondary to bladder outlet obstruction, conscious, normal, female Sprague-Dawley rats were investigated cystometrically before and after intracerebroventricular administration of RP 67,580, a selective antagonist of neurokinin (NK)-1 receptors and/or SR 48,968, a selective antagonist of NK-2 receptors. In normal rats, RP 67,580 or SR 48,968, at a dose of 2 nmol, caused no marked changes in cystometric parameters. Higher doses (up to 20 nmol) caused dose-dependent decreases in micturition pressure and increased bladder capacity, micturition volume and residual urine. A combination of the two drugs, each at a dose of 2 nmol, significantly decreased micturition pressure and increased bladder capacity. In rats with bladder outlet obstruction, the antagonists suppressed micturition dose-dependently, producing urinary retention in two out of eight rats already at a dose of 2 nmol. At a dose of 20 nmol, dribbling incontinence, due to urinary retention, was seen in five out of ten rats. A combination of the two drugs (2 nmol of each drug) caused urinary retention in three out of nine animals and significantly increased bladder capacity, micturition volume and residual volume. The results suggest that outflow obstruction in rats increases the effects of tachykinins in supraspinal structures involved in micturition, and that antagonism of supraspinal NK-receptors may depress the micturition reflex. Whether or not this implies that supraspinal NK-receptors can be targets for drugs aimed for inhibiting bladder hyperactivity in humans should be explored.

Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23.

Chronic lymphocytic leukaemia (B-CLL) is characterized by a progressive accumulation of B lymphocytes in blood and bone marrow and high concentrations of soluble CD23 and L-selectin are found in the serum of these patients. In this study lymphocytes from normal subjects and patients with B-CLL were allowed to undergo transendothelial migration across confluent layers of human umbilical vein endothelial cells. Lymphocytes in B-CLL samples showed an impaired capacity to migrate while the minor proportion of normal T cells was enriched by a mean of 2.5-fold in the transmigrated lymphocytes. In contrast, the ratio of B to T lymphocytes in normal preparations was unchanged in the transmigrated population. The expression of adhesion molecules on B-CLL lymphocytes before and after transendothelial migration was studied by flow cytometry which showed that 71 +/- 5% of L-selectin was lost from the surface of transmigrated lymphocytes. T and B cells from normal subjects also showed a major loss of L-selectin after transmigration. B-CLL lymphocytes and normal B cells expressed CD23 but this molecule was down-regulated following transendothelial migration, whereas the expression of VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with oxidized ATP, an irreversible inhibitor of P2Z/P2X7 purinoceptors, retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Our results show that B-CLL lymphocytes have impaired ability for transendothelial migration compared to normal peripheral blood lymphocytes. Moreover, transendothelial migration involves a universal loss of L-selectin and CD23 from lymphocytes which suggests that the high serum levels of soluble L-selectin and CD23 observed in B-CLL may be generated by shedding during the process of transendothelial migration.

Enhanced continent mechanism of tapered ileum by extramural support from pouch-abdominal wall: an experimental study in dogs.

PURPOSE: To construct a reliable continent tube, which is easy to catheterize, we created and evaluated a new continent cutaneous diversion using a tapered ileum combined with extramural support from the pouch-abdominal wall. MATERIALS AND METHODS: Six dogs underwent a procedure in which two ileal segments were tapered and anastomosed to the ileal pouch. One of the segments (continent) was placed between the back surface of the rectus muscle and the wall of the ileal pouch creating a continent tube. The other segment (control) was brought out into the abdominal skin directly without any support from the pouch-abdominal wall. Urodynamic and radiological studies were carried out postoperatively in all dogs. RESULTS: In the continent tubes, the maximum closure pressure with a full pouch was significantly higher than those with an empty pouch (p<0.01). The maximum closure pressures of the continent tubes were significantly higher than those of the control tubes when the pouch was empty (p<0.01). The leak point pressures of the continent tubes were significantly higher than those of the control tubes (p<0.001). In all dogs, the retrograde radiogram of the continent tubes showed perfect canalization without stenosis. CONCLUSION: This study suggests that the continent mechanism of tapered ileum can be greatly enhanced by fixing it between the abdominal and pouch walls, also allowing easy catheterization in contrast to the submucosal tunnel technique.