[Discussion of grading method of small opacity profusion of pneumoconiosis on CT scans and the corresponding reference images].

Objective: To explore the CT grading method of small opacity profusion of pneumoconiosis, and draw up the corresponding CT reference film. Methods: In December 2019, Three hundred thirty-seven cases of pneumoconosis and suspected pneumoconiosis were examined by chest radiography and Computed Tomography (CT) in the same period. According to Diagnosis of Occupational Pneumoconiosis (GBZ 70-2015) , small opacity profusion of pneumoconiosis in each zone of lung was divided. On CT scans, it was divided into 5 grades of 0, 0+, 1, 2 and 3. Grade 0 corresponded to Sub-grade 0/- and Sub-grade 0/0 of Grade 0 in chest radiograph. Grade 0+ was equivalent to Sub-grade 0/1 of Grade 0. Grade 1, 2, 3 were equivalent to Grade 1, 2 and 3, respectively (including each sub-grade) . The CT image quality of each zone of lung was divided into 1 to 4 levels. Results of level 4 were not included in statistical analyses.Based on the results of small opacity profusion in each zone of lung, consistency analysis was performed between chest radiograph and CT. The selection method of reference films was developed. Based on the types and grades of small opacity, the final reference films were determined. Results: There were 1877 zones of lung with CT image quality from level 1 to 3, including 335 in upper right, 319 in middle right, 284 in lower right, 334 in upper left, 320 in middle left and 286 in lower left. The Kappa values of small opacity profusion in upper right zone, upper left zone, left middle zone, and lower left zone were all between 0.4-0.75. In middle right zone and lower right zone, they were all above 0.75.Among all 6 zones of lung, the diagnostic concordance rates between CT and chest radiograph were all above 80%.The corresponding CT reference films were proposed, including type p and q in Grade 2 and 3, type r in Grade 2, type s and t in Grade 0+ to 3. Conclusion: The CT grading method for small opacity profusion of pneumoconiosis is feasible, and the application value of its reference films needs to be further verified.

LncRNA MALAT1 facilities high glucose induced endothelial to mesenchymal transition and fibrosis via targeting miR-145/ZEB2 axis.

OBJECTIVE: Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus (DM), but the pathophysiology of DN is complex and not fully understood. Renal tubal epithelial-mesenchymal transition (EMT) has been shown to be the critical mechanism of glomerulosclerosis and tubulointerstitial fibrosis. However, the precise mechanisms underlying EMT are not clear. MALAT1 was found induced by hyperglycemia in kidney but whether MALAT1 is involved in renal tubal EMT remains unknown. The objective of our study is to explore the role of MALAT1 in hyperglycemia-induced EMT and fibrosis. PATIENTS AND METHODS: We used db/db mouse and high glucose (HG)-stimulated HK-2 cells as in vivo and in vitro model of DN, respectively. qRT-PCR was used to measure levels of MALAT1 and miR-145. In addition, we validated interactions of MALAT1-miR-145 and miR-145-ZEB2 by dual luciferase reporter assays. Western blot was used to examine expressions of proteins involved in EMT and fibrosis. RESULTS: MALAT1 was upregulated while miR-145 was downregulated in renal tissues of db/db mice. Consistently, hyperglycemia significantly increased the level of MALAT1 but decreased miR-145 expression in a time-dependent manner in HK-2 cells. Furthermore, miR-145 binds to both MALAT1 and ZEB2. Knockdown MALAT1 or ZEB2 inhibited HG-induced EMT and fibrosis, similar to miR-145 overexpression. CONCLUSIONS: Our study is the first to show that MALAT1 and miR-145 regulate HG-induced EMT and fibrosis. Mechanistically, MALAT1 functions as a sponge RNA for miR-145 to derepress the expression of target gene ZEB2, thereby inducing EMT and fibrosis. These results provide a novel potential target for DN therapy in the future.

Protective effect of Lycium barbarum polysaccharides against high-fat diet-induced renal injury and lipid deposition in rat kidneys.

This study aimed to explore the protective effect of Lycium barbarum polysaccharides (LBPs) against hyperlipidemia and lipid-induced renal injury in a rat model. Male Sprague-Dawley rats (n=30) were randomly divided into three equal groups: a control group (fed a regular diet) and two experimental groups (fed a high-fat diet). By feeding rats a high-fat diet for 12 weeks, an animal model of hyperlipidemia was established, after which one experimental group received oral LBPs at a dose of 300 mg/kg per day. Blood lipids, renal function, and urinary proteins were measured after 12 weeks. Changes in renal pathology and expression levels of sterol regulatory element binding transcription factor 1 (SREBP-1), interleukin-6 (IL- 6), tumor necrosis factor-alpha (TNF-α), AMP-activated protein kinase (AMPK) were determined. Rats with hyperlipidemia induced by a high-fat diet showed increases in blood lipids and blood urea nitrogen, serum creatinine, and urinary protein, as well as increases in renal levels of SREBP-1, TNF-α, and IL-6 and decreases in renal levels of adiponectin and AMPK. Administration of LBPs restored blood lipid, blood urea nitrogen, serum creatinine, and urinary protein levels, downregulated renal levels of SREBP-1, TNF-α, and IL-6, and upregulated renal levels of adiponectin and AMPK. These results indicate that LBPs may mediate lipid metabolism, enhance anti-inflammatory responses, and ameliorate renal injury caused by lipid metabolism isorders in a rat model of hyperlipidemia.

MicroRNA-1294 targets HOXA9 and has a tumor suppressive role in osteosarcoma.

OBJECTIVE: MicroRNA-1294 (miR-1294) was reported to act as a tumor suppressor in several cancers. However, the biological function of miR-1294 in osteosarcoma (OS) has not been investigated. We, therefore, investigated the clinical significance and underlying mechanisms of miR-1294 in OS. PATIENTS AND METHODS: Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) was conducted to detect the levels of miR-1294. Targets of miR-1294 were validated by luciferase reporter assay and Western blot. In vitro functional assays were performed to investigate the effects of miR-217 on cell proliferation and invasion. RESULTS: We found miR-1294 was downregulated in OS tissues and cell lines. Downregulation of miR-1294 has a significant negative impact on the overall survival of OS patients. Overexpression of miR-1294 suppresses OS cell proliferation and invasion in vitro. Then, luciferase reporter assay validated Homeobox A9 (HOXA9) was a downstream target of miR-1294. Expression patterns of miR-1294 were inversely correlated with HOXA9 in OS tissues, strengthening the findings from the luciferase reporter assay. Further functional assays revealed that overexpression of HOXA9 could reverse the inhibition effects of miR-1294 on cell proliferation and invasion. CONCLUSIONS: These results suggested miR-1294 functions as a tumor suppressor in OS progression by targeting HOXA9.

[Analysis of risk factors for acute lung injury/acute respiratory distress syndrome after esophagectomy].

OBJECTIVE: To explore the incidence and risk factors for the acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) after resection of esophageal carcinoma. METHODS: We retrospectively analyzed 422 consecutive patients admitted to the Department of Critical Care Medicine with esophageal carcinoma undergoing esophagectomy from January 2010 to December 2016 in Peking University Cancer Hospital. ALI/ARDS were diagnosed, the patients were divided into ALI/ARDS group and control group without ALI/ARDS, the differences of clinical features were contrasted between the two groups, and the multivariate Logistic regression modeling was used to identify the independent risk factors for ALI/ARDS. RESULTS: In the study, 41 ALI/ARDS cases were diagnosed, making up 9.7% (41/422) of all the enrolled patients undergoing esophagectomy. Comparisons of the ALI/ARDS group and the control group indicated significant statistical differences in the average length of their hospital stay [(18.9±9.7) d vs. (14.8±3.6) d, P=0.011], the proportion of the patients who needed mechanical ventilation support [51.2% (21/41) vs. 9.4% (36/381), P<0.001] and in-hospital mortality [31.7% (13/41) vs. 5.0% (19/381), P<0.001]. Univariate analysis showed significant differences between the patients with ALI/ARDS and without ALI/ARDS in smoking history (P=0.064), preoperative forced expiratory volume in one second/forced vital capacity (FEV1/FVC) (P=0.020), diffusing capacity of the lung for carbon monoxide (DLCO) (P=0.011), body weight index (BMI) (P=0.044), American Society of Anesthesiologists (ASA) physical status classification (P=0.049) and one lung ventilation duration (P=0.008), while multivariate Logistic regression analysis indicated that preoperative FEV1/FVC (OR=1.053, P=0.016, 95%CI 1.010-1.098), ASA physical status classification (OR=2.392, P=0.033, 95%CI 1.073-5.335) and one lung ventilation duration (OR=0.994, P=0.028, 95%CI 0.989-0.999) were the independent risk factors for ALI/ARDS after esophagectomy. CONCLUSION: ALI/ARDS was a serious complication in patients undergoing esophagectomy associated with increment in length of hospital stay and in-hospital mortality. Multivariate Logistic regression analysis indicated that preoperative FEV1/FVC, ASA classification and one lung ventilation duration were the independent risk factors for ALI/ARDS after esophagectomy. Carefully assessing the patient before operation, shortening one lung ventilation duration were the key points in preventing ALI/ARDS after esophagectomy.

Systemic gene delivery transduces the enteric nervous system of guinea pigs and cynomolgus macaques.

Characterization of adeno-associated viral vector (AAV) mediated gene delivery to the enteric nervous system (ENS) was recently described in mice and rats. In these proof-of-concept experiments, we show that intravenous injections of clinically relevant AAVs can transduce the ENS in guinea pigs and non-human primates. Neonatal guinea pigs were given intravenous injections of either AAV8 or AAV9 vectors that contained a green fluorescent protein (GFP) expression cassette or phosphate-buffered saline. Piglets were euthanized three weeks post injection and tissues were harvested for immunofluorescent analysis. GFP expression was detected in myenteric and submucosal neurons along the length of the gastrointestinal tract in AAV8 injected guinea pigs. GFP-positive neurons were found in dorsal motor nucleus of the vagus and dorsal root ganglia. Less transduction occurred in AAV9-treated tissues. Gastrointestinal tissues were analyzed from young cynomolgus macaques that received systemic injection of AAV9 GFP. GFP expression was detected in myenteric neurons of the stomach, small and large intestine. These data demonstrate that ENS gene delivery translates to larger species. This work develops tools for the field of neurogastroenterology to explore gut physiology and anatomy using emerging technologies such as optogenetics and gene editing. It also provides a basis to develop novel therapies for chronic gut disorders.

TRIM59 is a key regulator of growth and migration inrenal cell carcinoma.

Renal cell carcinoma (RCC) is the most common renal neoplasms and metastatic is common. Previous data have shown that the tripartite motif (TRIM) family proteinswere implicated in human tumoriogenesis. In this study, we aimed to investigate the role of TRIM59 in the cell growth and migration in RCC. The expression of TRIM59 in human RCC tissues was initially examined by qRT-PCR. Alentivirus-based shRNA against TRIM59 (Lv-shTRIM59) was constructed. The effects of TRIM59 knockdown on cell proliferation were examined by in vitro MTT assay, colony formation assay and in vivo a mouse xenograft model of RCC. Cell migration and invasion after knockdown of TRIM59 were also examined by transwell assay. Our data showed that the mRNA level of TRIM59 in cancerous tissues was 2-fold increased as compared with non-cancerous tissues. Knockdown of TRIM59 in a RCC cell line 786-O significantly slowed down cell proliferative rate and decreased both the colony number and sizes. In the mouse model, knockdown of TRIM59 consistently inhibited tumor growth in vivo. Moreover, it was shown that cell migration and invasion were suppressed by 68% and 50%, respectively in TRIM59-depleted 786-O cells. Our data suggest that TRIM59 may serve as a pro-oncogenic protein in promoting the progression of RCC. Knockdown of TRIM59 may be a promising strategy concerning the early detection and treatment of RCC.

Pivotal role of microRNA-9 in osteosarcoma tumorigenesis and tumor progression.

MicroRNA-9 (miR-9) has a well-established role in various tumors; the clinical significance and potential mechanism of miR-9 in human osteosarcoma (OS) has not been elucidated. The aim of this study was to investigate the mechanism and role of miR-9 expression in osteosarcoma cells. miR-9 expression in the OS cell line MG-63 and OS tissues was compared to that in a human osteoblastic cell line (hFOB 1.19) and adjacent normal tissues, respectively, by reverse transcriptase-polymerase chain reaction. miR-9 expression was downregulated by introducing small interfering RNA against miR-9 (si-miR-9) into the cells, and the proliferative, migratory, and invasive capacities of si-miR-9-transfected MG-63 cells were compared to those of control MG-63 cells. miR-9 was significantly upregulated in OS tissues and cell lines compared to the corresponding non-cancerous bone tissues (P < 0.05) and human osteoblastic cell line (P < 0.05), respectively. Upregulated miR-9 expression was also associated with increased cell proliferation (P < 0.05), migration (P < 0.05), and invasion (P < 0.05), and decreased apoptotic ability (P < 0.05). These results suggest that miR-9 may play a pivotal role in tumorigenesis and tumor progression in osteosarcoma.

[Effect of 2-methoxyestradiol combined with quercetin on prostate cancer in vitro].

OBJECTIVE: To detect the effects of quercetin (Que) combined with 2-methoxyestradiol (2-ME) on the proliferation of androgen-dependent LNCaP human prostate cancer cells line and androgen-independent PC-3 human prostate cancer cells line, and to evaluate the antitumor effects of different combos of the two drugs. METHODS: After LNCaP and PC-3 cells were treated with different concentration of quercetin (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 µmol/L) or 2-ME (0, 0.312 5, 0.625, 1.25, 2.5, 5, 10 µmol/L) for 48 h, the inhibitory rates of cell growth were tested using trypan blue staining method respectively. Then the concentration-effect curves were drawn and IC(50) values were calculated. According to the fitted dose-effect curves and IC(50) values, appropriate concentrations of quercetin and 2-ME were selected to compose 16 different combos. Then cells were treated with different combos of Que and 2-ME for 48 h, and then the growth inhibitory rates of cell growth were detected. According to the equation and median-effect principle, the CI values of 16 different combos of Que and 2-ME were calculated to evaluate their antitumor effects. RESULTS: The inhibition rate of LNCaP or PC-3 cell growth treated with varying doses of quercetin or 2-ME alone showed a dose-dependent increase respectively. The IC(50) values of quercetin and 2-ME were 23.29 µmol/L and 1.89 µmol/L for LNCaP cells; and 22.12 µmol/L and 1.74 µmol/L for PC-3 cells respectively. After treated with 16 combos of Que (5, 10, 20, 40 µmol/L) and 2-ME (0.5, 1, 3, 5 µmol/L) for 48 h, for LNCaP cells, lower dose of Que (5 and 10 µmol/L) with higher dose of 2-ME (3 and 5 µmol/L) showed synergistic activity, whereas for PC-3 cells, besides the above combination of Que 10 µmol/L and 2-ME 3 µmol/L, higher dose of quercetin (20 and 40 µmol/L) with higher dose of 2-ME (3 and 5 µmol/L) also showed synergistic activity. CONCLUSIONS: Both quercetin and 2-methoxyestradiol could inhibit the growth of LNCaP and PC-3 human prostate cancer cells in a dose dependent manner. We confirmed that combinations of quercetin and 2-ME at appropriate concentrations have the potential for synergetic antiproliferative activity in vitro.

Potential gains in life expectancy from reducing heart disease, cancer, Alzheimer's disease, kidney disease or HIV/AIDS as major causes of death in the USA.

OBJECTIVES: Potential gains in life expectancy (PGLEs) that give proper consideration to competing risks are an effective indicator for measuring the impact of multiple causes of death on a defined population. This study aimed to assess PGLE by hypothetically reducing the major causes of death in the USA from 2001 to 2008. STUDY DESIGN: PGLEs due to the reduction and elimination of heart disease, cancer, Alzheimer's disease, kidney disease or human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) were calculated by age, gender and race. METHODS: Age-specific mortality rates for the above diseases from the National Center for Health Statistics were used, and multiple decremental life tables were constructed to compute the corresponding PGLEs. RESULTS: PGLEs due to the elimination of heart disease, cancer or HIV/AIDS decreased from 2001 to 2008, but PGLEs due to the elimination of Alzheimer's disease or kidney disease increased over time. For heart disease, PGLE in 2001-2008 for all races was 2.78-2.15 for females vs 2.41-2.06 for males. For cancer, PGLE in 2001-2008 for all races was 2.97-2.81 for females vs 3.02-2.85 for males. HIV/AIDS has a greater impact on people of working age, whereas Alzheimer's disease has a greater impact on the elderly population. To compare the impacts of these diseases on life expectancy, partial multiple decremental life tables were constructed, and PGLEs were computed by a partial reduction or complete elimination of various causes of death for the entire life span as well as for certain working ages. CONCLUSION: This study outlined a picture of how each category of diseases could affect life expectancy in the US population by age, race or sex. The findings may assist in evaluating current public health improvements, and also provide useful information for directing future research and disease control programmes.

Neurogenic mucosal bicarbonate secretion in guinea pig duodenum.

BACKGROUND AND PURPOSE: To test a hypothesis that: (i) duodenal pH and osmolarity are individually controlled at constant set points by negative feedback control centred in the enteric nervous system (ENS); (ii) the purinergic P2Y(1) receptor subtype is expressed by non-cholinergic secretomotor/vasodilator neurons, which represent the final common excitatory pathway from the ENS to the bicarbonate secretory glands. EXPERIMENTAL APPROACH: Ussing chamber and pH-stat methods investigated involvement of the P2Y(1) receptor in neurogenic stimulation of mucosal bicarbonate (HCO(3)(-)) secretion in guinea pig duodenum. KEY RESULTS: ATP increased HCO(3)(-) secretion with an EC(50) of 160 nM. MRS2179, a selective P2Y(1) purinergic receptor antagonist, suppressed ATP-evoked HCO(3)(-) secretion by 47% and Cl(-) secretion by 63%. Enteric neuronal blockade by tetrodotoxin or exposure to a selective vasoactive intestinal peptide (VIP, VPAC(1)) receptor antagonist suppressed ATP-evoked HCO(3)(-) secretion by 61 and 41%, respectively, and Cl- by 97 and 70% respectively. Pretreatment with the muscarinic antagonist, scopolamine did not alter ATP-evoked HCO3(-) or Cl(-) secretion. CONCLUSION AND IMPLICATIONS: Whereas acid directly stimulates the mucosa to release ATP and stimulate HCO(3)(-) secretion in a cytoprotective manner, neurogenically evoked HCO(3)(-) secretion accounts for feedback control of optimal luminal pH for digestion. ATP stimulates duodenal HCO(3)(-) secretion through an excitatory action at purinergic P2Y(1) receptors on neurons in the submucosal division of the ENS. Stimulation of the VIPergic non-cholinergic secretomotor/vasodilator neurons, which are one of three classes of secretomotor neurons, accounts for most, if not all, of the neurogenic secretory response evoked by ATP.

Effect of processing route on the surface properties of amorphous indomethacin measured by inverse gas chromatography.

The aim of this study was to investigate the effect of processing route (i.e., quench cooling and ball milling) on the surface energy heterogeneity and surface chemistry of indomethacin (IMC). Recently developed inverse gas chromatography (IGC) methodology at finite concentrations was employed to determine the surface energy distributions of crystalline, quench cooled and milled IMC samples. Surface properties of crystalline and processed IMC were measurably different as determined by the IGC and other conventional characterization techniques: differential scanning calorimetry and powder X-ray diffraction. Quench cooled IMC was in fully amorphous form. Milled IMC showed no amorphous character by calorimetric or X-ray diffraction studies. It was demonstrated that both processed IMC samples were energetically more active than the crystalline IMC. In particular, milled IMC exhibited a relatively higher dispersive surface energy and higher surface basicity (electron donor capability). This may be attributed to the creation of surface defect sites or exposure of higher energy crystal facets during the milling process. This study confirms that processing route has notable influence on the surface energy distribution and surface acid-base character. IGC was demonstrated as a powerful technique for investigating surface properties of real-world, heterogeneous pharmaceutical materials.

Analysis of 13 cases of venous compromise in 178 radial forearm free flaps for intraoral reconstruction.

The purpose of this study was to analyse the causes of venous compromise and flap failure in radial forearm free flap (RFFF) surgery for intraoral reconstruction. One hundred seventy-eight RFFF reconstructions were reviewed retrospectively for intraoral defects. Of the 13 flaps with venous obstruction, 9 flaps were salvaged, and 4 were lost, with a salvage rate of 69.2%. Eleven venous occlusions occurred within the first 72h. The main reasons for venous failure were mechanical obstruction or technical errors due to inadequate pedicle length and geometry, inadequate venous drainage, compression and kinking of the vein. The main cause of failure for oropharynx reconstruction was unrecognized vascular events due to the lack of reliable monitoring for buried flap. Oozing of dusky blood from the flap margin may be directly related to venous congestion in the early postoperative period and a late indication of a change in skin colour. In conclusion, a thorough operative plan, including carefully selected drainage vein for the flap and recipient vessels, adequate pedicle length and geometry, precise surgical technique, avoidance of haematoma, and expert monitoring of buried flaps may improve the success rate of RFFF transfer in intraoral reconstruction.

Differential actions of urocortins on neurons of the myenteric division of the enteric nervous system in guinea pig distal colon.

BACKGROUND AND PURPOSE: Urocortins (Ucns) 1, 2 and 3 are corticotropin-releasing factor (CRF)-related neuropeptides and may be involved in neural regulation of colonic motor functions. Nevertheless, details of the neural mechanism of action for Ucns have been unclear. We have, here, tested the hypothesis that Ucns act in the enteric nervous system (ENS) to influence colonic motor behaviour. EXPERIMENTAL APPROACH: We used intracellular recording with 'sharp' microelectrodes, followed by intraneuronal injection of biocytin, and immunohistochemical localization of CRF(1) and CRF(2) receptors in guinea pig colonic tissue. KEY RESULTS: Application of Ucn1 depolarized membrane potentials and elevated excitability in 58% of AH-type and 60% of S-type colonic myenteric neurons. In most of the neurons tested, depolarizing responses evoked by Ucn-1 were suppressed by the CRF(1) receptor antagonist NBI 27914, but were unaffected by the CRF(2) receptor antagonist antisauvagine-30. The selective CRF(2) receptor agonists, Ucn2 and Ucn3, evoked depolarizing responses in 12 and 8% of the AH-type myenteric neurons, respectively, and had no effect on S-type neurons. Antisauvagine-30, but not NBI 27914, suppressed these Ucn2- and Ucn3-evoked responses. Immunohistochemical staining identified CRF(1) as the predominant CRF receptor subtype expressed by ganglion cell somas, while CRF(2)-immunoreactive neuronal somas were sparse. Ucns did not affect excitatory synaptic transmission in the ENS. CONCLUSIONS AND IMPLICATIONS: The results suggest that Ucns act as neuromodulators to influence myenteric neuronal excitability. The excitatory action of Ucn1 in myenteric neurons was primarily at CRF(1) receptors, and the excitatory action of Ucn2 and Ucn3 was at CRF(2) receptors.

Neurogastroenterology of tegaserod (HTF 919) in the submucosal division of the guinea-pig and human enteric nervous system.

Actions of the 5-HT(4) serotonergic receptor partial agonist, tegaserod, were investigated on mucosal secretion in the guinea-pig and human small intestine and on electrophysiological behaviour of secretomotor neurons in the guinea-pig small intestinal submucosal plexus. Expression of 5-HT(4) receptor protein and immunohistochemical localization of the 5-HT(4) receptor in the submucosal plexus in relation to expression and localization of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter were determined for the enteric nervous system of human and guinea-pig small intestine. Immunoreactivity for the 5-HT(4) receptor was expressed as ring-like fluorescence surrounding the perimeter of the neuronal cell bodies and co-localized with the vesicular ACh transporter. Exposure of mucosal/submucosal preparations to tegaserod in Ussing chambers evoked increases in mucosal secretion reflected by stimulation of short-circuit current. Stimulation of secretion had a relative high EC(50) of 28.1 +/- 1.3 mumol L(-1), was resistant to neural blockade and appeared to be a direct action on the secretory epithelium. Tegaserod acted at presynaptic 5-HT(4) receptors to facilitate the release of ACh at nicotinic synapses on secretomotor neurons in the submucosal plexus. The 5-HT(2B) receptor subtype was not involved in actions at nicotinic synapses or stimulation of secretion.

Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome.

The availability of huge amounts of rice genome sequence now permits large-scale analysis of the structure and molecular characteristics of the previously identified transposase-encoding Rim2 (also called Hipa) element, which is transcriptionally activated by infection with the fungal pathogen Magnaporthe grisea and by treatment with the corresponding fungal elicitor. Based on genomic cloning and data mining from 230 Mb of rice genome sequence, 347 Rim2 elements, with an average size of 5.8 kb, were identified. This indicates that an estimated total of 600-700 Rim2 elements are present in the whole genome. Rim2 insertions occur non-randomly on the chromosomes, as visualized by fluorescence in situ hybridization. The elements harbor 16-bp terminal inverted repeats with the core sequence CACTG, 16-bp sub-terminal repeats, internal variable regions, 3-bp target sequence duplications in the flanking regions, and genes coding for Rim2 proteins (the putative transposase) and hydroxyproline-rich glycoproteins. High levels of insertion into genic regions are observed for members of this family, and the transposition history of the family can be deduced from the high level of shared sequences and analysis of repeat target sites of the elements. Phylogenetic analysis indicates that the putative RIM2 proteins fall into a subgroup distinct from the TNP2-like subgroup of transposases. Southern hybridization with genomic DNA from monocotyledonous and dicotyledonous plants demonstrates that the RIM2-coding sequence is unique to the Oryza genome. Our results demonstrate that the Rim2 elements from rice belong to a distinct superfamily of CACTA-like elements with evolutionary diversity.

Oxytocin mediates stress-induced analgesia in adult mice.

As a neurohormone and as a neurotransmitter, oxytocin has been implicated in the stress response. Descending oxytocin-containing fibres project to the dorsal horn of the spinal cord, an area important for processing nociceptive inputs. Here we tested the hypothesis that oxytocin plays a role in stress-induced analgesia and modulates spinal sensory transmission. Mice lacking oxytocin exhibited significantly reduced stress-induced antinociception following both cold-swim (10 degrees C, 3 min) and restraint stress (30 min). In contrast, the mice exhibited normal behavioural responses to thermal and mechanical noxious stimuli and morphine-induced antinociception. In wild-type mice, intrathecal injection of the oxytocin antagonist dOVT (200 microM in 5 microl) significantly attenuated antinociception induced by cold-swim. Immunocytochemical staining revealed that, in the mouse, oxytocin-containing neurones in the paraventricular nucleus of the hypothalamus are activated by stress. Furthermore, oxytocin-containing fibres were present in the dorsal horn of the spinal cord. To test whether descending oxytocin-containing fibres could alter nociceptive transmission, we performed intracellular recordings of dorsal horn neurones in spinal slices from adult mice. Bath application of oxytocin (1 and 10 microM) inhibited excitatory postsynaptic potentials (EPSPs) evoked by dorsal root stimulation. This effect was reversed by the oxytocin antagonist dOVT (1 microM). Whole-cell recordings of dorsal horn neurones in postnatal rat slices revealed that the effect of oxytocin could be blocked by the addition of GTP-gamma-S to the recording pipette, suggesting activation of postsynaptic oxytocin receptors. We conclude that oxytocin is important for both cold-swim and restraint stress-induced antinociception, acting by inhibiting glutamatergic spinal sensory transmission.

Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism.

In the spinal cord dorsal horn, excitatory sensory fibers terminate adjacent to interneuron terminals. Here, we show that kainate (KA) receptor activation triggered action potential-independent release of GABA and glycine from dorsal horn interneurons. This release was transient, because KA receptors desensitized, and it required Na+ entry and Ca2+ channel activation. KA modulated evoked inhibitory transmission in a dose-dependent, biphasic manner, with suppression being more prominent. In recordings from isolated neuron pairs, this suppression required GABA(B) receptor activation, suggesting that KA-triggered GABA release activated presynaptic GABA(B) autoreceptors. Finally, glutamate released from sensory fibers caused a KA and GABA(B) receptor-dependent suppression of inhibitory transmission in spinal slices. Thus, we show how presynaptic KA receptors are linked to changes in GABA/glycine release and highlight a novel role for these receptors in regulating sensory transmission.

Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis.

In cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)-delta-cadinene, forming 8-hydroxy-(+)-delta-cadinene. This P450 mono-oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)-delta-cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.

Genetic enhancement of inflammatory pain by forebrain NR2B overexpression.

N-methyl-D-aspartate (NMDA) receptors contribute to many brain functions. We studied the effect of forebrain-targeted overexpression of the NMDA receptor subunit NR2B on the response of mice to tissue injury and inflammation. Transgenic mice exhibited prominent NR2B expression and enhanced NMDA receptor-mediated synaptic responses in two pain-related forebrain areas, the anterior cingulate cortex and insular cortex, but not in the spinal cord. Although transgenic and wild type mice were indistinguishable in tests of acute pain, transgenic mice exhibited enhanced responsiveness to peripheral injection of two inflammatory stimuli, formalin and complete Freund's adjuvant. Genetic modification of forebrain NMDA receptors can therefore influence pain perception, which suggests that forebrain-selective NMDA receptor antagonists, including NR2B-selective agents, may be useful analgesics for persistent pain.