Fibre type specificity of haem oxygenase-1 induction in rat skeletal muscle.

The expression of the inducible haem oxygenase (HO-1) gene was examined in different skeletal muscles. Rats were treated with haemin and a time course of HO-1 mRNA expression was determined in soleus and extensor digitorum longus (EDL) muscles. Fibre type composition and tissue myoglobin content were also measured. We found that HO-1 mRNA expression markedly increased in soleus (type I fibres) muscle but was only slightly affected in EDL (type II fibres). HO-1 expression directly correlated with both percentage of red fibres and tissue myoglobin. These data demonstrate that HO-1 gene expression follows a fibre type-specific pattern which might indicate an important role for this protein in the maintenance of skeletal muscle function.     

Effects of protoporphyrins on production of nitric oxide and expression of vascular endothelial growth factor in vascular smooth muscle cells and macrophages

Heme oxygenase-1 (HO-1), an inducible enzyme degrading heme to biliverdin, iron and carbon monoxide, is involved in regulation of inflammation and angiogenesis. Tin protoporphyrin (SnPPIX) and zinc protoporphyrin (ZnPPIX) are commonly used as competitive inhibitors of HO-1. We aimed to compare the effects of SnPPIX and ZnPPIX on the production of vascular endothelial growth factor (VEGF), activity of inducible nitric oxide synthase (iNOS) and cell viability. All experiments were performed on rat vascular smooth muscle cells and murine RAW264.7 macrophages treated with 3-10 microM protoporphyrins. Some cells were additionally stimulated with IL-1beta or with lipopolysaccharide. After a 24 h incubation period SnPPIX and ZnPPIX significantly reduced the generation of VEGF in vascular smooth muscle cells and RAW264.7, both in resting and stimulated cells. The inhibitory potentials of both protoporphyrins on VEGF synthesis were very similar. In contrast, analysis of iNOS activity revealed that results obtained with different HO-1 inhibitors are discrepant. Generation of nitric oxide by iNOS was significantly increased by SnPPIX but strongly decreased by ZnPPIX. Similar differences were observed when cell viability was compared. SnPPIX improved the cell survival rate, whereas the same doses of ZnPPIX exerted some cytotoxic effects. In summary, SnPPIX and ZnPPIX can be used as HO-1 inhibitors in some experimental models. However, these compounds produce also HO-independent effects, which can make the interpretation of experiments very uncertain. Thus the involvement of the HO-1 pathway should be always confirmed by more specific methods.     

内毒素引起的乳鼠心肌细胞血红素加氧酶—1基因的表达

为了探讨在内毒素作用下的乳鼠心肌细胞（neonatal rat cardiomyocytes,NRCMs）血红素加氧酶－1（heme oxygenase-1,HO-1）基因的表达及其在细胞损伤中的作用,分别用10、30及50μg/ml的脂多糖（lipopolysaccharide,LPS）,10μg/ml LPS 10μmol/ml锌原卟啉Ⅸ（Zn-protoporphyrin-Ⅸ,ZnPPⅨ）和单纯10μmol/ml ZnPPⅨ与培养的NRCMs共同孵育6h,以及10μg/ml LPS与NRCMs共同孵育9h和18h。分别观察细胞HO－1 mRNA表达、MDA含量、LDH释放量与台盼蓝摄取率的变化。结果显示,同样与细胞孵育6h,LPS10μg/ml时HO－1 mRNA表达比对照组增加81.2％,30μg/ml时表达量增加126.3％,50μg/ml时表达量增加92.8％;LPS为10μg/ml时,孵育9h后HO－1 mRNA的表达量比对照组增加93.6％,孵育18h后一增加105.8％。LPS30、50μg/ml,10μg/ml LPS＋10μmol/ml ZnPPⅨ与细胞孵育6h及LPS 10μg/ml孵育18h后,细胞MDA含量、LDH释放量与台盼蓝摄取率明显增加（P＜0.01）;单纯10μg/ml LPS与单纯10μmol/ml ZnPPⅨ孵育6h后,上述指标均无明显升高。结果表明,LPS可诱导NRCMs HO－1 mRNA的表达,且在较低LPS剂量范围内具有时间依赖性和浓度依赖性;NRCMs HO-1 mRNA的表达可减低LPS引起的细胞损伤,这可能是细胞产生的一种自身保护性反应。     

SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis

? The SOS signaling pathway plays an important role in plant salt tolerance. However, little is known about how the SOS pathway modulates organ development in response to salt stress. Here, the involvement of SOS signaling in NaCl-induced lateral root (LR) development in Arabidopsis was assessed. ? Wild-type and sos3-1 mutant seedlings on iso-osmotic concentrations of NaCl and mannitol were analyzed. The marker lines for auxin accumulation, auxin transport, cell division activity and stem cells were also examined. ? The results showed that ionic effect alleviates the inhibitory effects of osmotic stress on LR development. LR development of the sos3-1 mutant showed increased sensitivity specifically to low salt. Under low-salt conditions, auxin in cotyledons and LR primordia (LRP) of the sos3-1 mutant was markedly reduced. Decreases in auxin polar transport of mutant roots may cause insufficient auxin supply, resulting in defects not only in LR initiation but also in cell division activity in LRP. ? Our data uncover a novel role of the SOS3 gene in modulation of LR developmental plasticity and adaptation in response to low salt stress, and reveal a new mechanism for plants to sense and adapt to small changes of salt.     

Isolation and Functional Validation of Salinity and Osmotic Stress Inducible Promoter from the Maize Type-II H+-Pyrophosphatase Gene by Deletion Analysis in Transgenic Tobacco Plants

Salinity and drought severely affect both plant growth and productivity, making the isolation and characterization of salinity- or drought-inducible promoters suitable for genetic improvement of crop resistance highly desirable. In this study, a 1468-bp sequence upstream of the translation initiation codon ATG of the promoter for ZmGAPP (maize Type-II H⁺-pyrophosphatase gene) was cloned. Nine 5´ deletion fragments (D1–D9) of different lengths of the ZmGAPP promoter were fused with the GUS reporter and translocated into tobacco. The deletion analysis showed that fragments D1–D8 responded well to NaCl and PEG stresses, whereas fragment D9 and CaMV 35S did not. The D8 segment (219 bp; -219 to -1 bp) exhibited the highest promoter activity of all tissues, with the exception of petals among the D1–D9 transgenic tobacco, which corresponds to about 10% and 25% of CaMV 35S under normal and NaCl or PEG stress conditions, respectively. As such, the D8 segment may confer strong gene expression in a salinity and osmotic stress inducible manner. A 71-bp segment (-219 to -148 bp) was considered as the key region regulating ZmGAPP response to NaCl or PEG stress, as transient transformation assays demonstrated that the 71-bp sequence was sufficient for the salinity or osmotic stress response. These results enhance our understanding of the molecular mechanisms regulating ZmGAPP expression, and that the D8 promoter would be an ideal candidate for moderating expression of drought and salinity response genes in transgenic plants.     

Understanding Saline and Osmotic Tolerance of Populus euphratica Suspended Cells

Tolerance of Populus euphratica suspended cells to ionic and osmotic stresses implemented respectively by NaCl and PEG (6000) was characterized by monitoring cell growth, morphological features, ion compartmentation and polypeptide patterns. The cells grew and proliferated when submitted to stresses of 137 mM NaCl or 250 g l⁻¹ PEG, and survived at 308 mM of NaCl, showing tolerance to saline and particularly osmotic stress. They were resistant to plasmolysis and had dense cytoplasms, large nuclei and nucleoli, and evident cytoplasmic strands under high saline and osmotic stress. The sequestration of Cl⁻ into the vacuoles was observed in the cells stressed with 137 and 223 mM NaCl. The cellular protein profile was modified by high salt and osmotic stress and showed 28 kDa polypeptides up-regulated by both NaCl and PEG, and 66 and 25 kDa polypeptides up-regulated only by high NaCl stress. The salt tolerance of P. euphratica cells might be related to their capacity of adapting to higher osmotic stress by maintaining cell integrity, sequestrating Cl⁻ into vacuoles and modulating polypeptides that reflect cellular metabolic adaptations.     

Interaction between HY1 and H2O2 in auxin-induced lateral root formation in Arabidopsis

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H₂O₂) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H₂O₂ in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H₂O₂ generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H₂O₂. This conclusion was supported by analyzing the removal of H₂O₂ with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H₂O₂-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H₂O₂ generation, since Znpp inhibition of HY1 blocked the NAA-induced H₂O₂ production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H₂O₂ production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H₂O₂ production and LR formation, both of which were decreased by DMTU. Moreover, H₂O₂ and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H₂O₂ and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H₂O₂ existing in auxin-induced LR formation in Arabidopsis.     

The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.     

Differential responses of primary and lateral roots to indole-3-acetic acid,indole-3-butyric acid,and 1-naphthaleneacetic acid in maize seedlings

The role of auxins on root system architecture was studied by applying indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA) to maize roots and analysing the main processes involved in root development: primary root (PR) elongation, lateral root (LR) formation, and LR root elongation. We found that these effects were not dependent only on concentration, but also on the type of auxin applied. We also studied temporal changes in auxin inhibition of PR elongation. These temporal changes were analysed calculating the elongation ratio between two consecutive one day periods after auxin application. It was observed that a reduction in root elongation was also dependent on the type of auxin applied and its concentration. The inhibitory effect of IBA and IAA decreased on the second day, and the ratio also increased with the concentration. In contrast, NAA increased root elongation inhibition with time. Indeed, the ratio decreased as the NAA concentration increased. Regarding LR formation, we observed that external auxin increased only LR formation in certain zones of the PR. Finally, comparison of inhibition elongation associated with auxin in the LR and PR clearly demonstrates that PR elongation was more sensitive to auxin than LR elongation.     

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.     

Osmotic stress- and indole-3-butyric acid-induced NO generation are partially distinct processes in root growth and development in Pisum sativum

In this work, the effects of osmotic stress and exogenous auxin (indole-3-butyric acid, IBA) on root morphology and nitric oxide (NO) generation in roots were compared in pea plants. Five-day-old plants were treated with 0, 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹  M IBA or with PEG 6000 at concentrations that determined 0, 50, 100, 200 or 400 mOsm in the medium, during 5 days. NO generation was examined by in situ and in vivo fluorescence method. Increasing concentrations of PEG as well as IBA resulted in shortening of primary root (PR), enhancement of lateral root (LR) number and significant increase of NO generation. Time-dependent investigations revealed that in the case of IBA treatments, the LR number increased in parallel with an intensified NO generation, while elongation of PR was not followed by changes in NO levels. Under osmotic stress, the time curve of NO development was distinct compared with that of IBA-treated roots, because significantly, the appearance of lateral initials was preceded by a transient burst of NO. This early phase of NO generation under osmotic stress was clearly distinguishable from that which accompanied LR initiation. It is concluded that osmotic stress and the presence of exogenous auxin resulted in partly similar root architecture but different time courses of NO synthesis. We suppose that the early phase of NO generation may fulfill a role in the osmotic stress-induced signalization process leading to the modification of root morphology.     

Differential effect of NaCl and polyethylene glycol on the ultrastructure of chloroplasts in rice seedlings

Ionic and osmotic effects of salinity on the ultrastructure of chloroplasts in salt-treated rice seedlings were investigated. After rice seedlings were grown in hydroponic culture for three weeks, they were treated with NaCl and polyethylene glycol (PEG) 4000 both at a water potential of -1.0 MPa for 3 days. The most notable difference in ultrastructural change between NaCl and PEG treatment was observed in the damage in chloroplast membranes. NaCl induced swelling of thylakoids and caused only a slight destruction of the chloroplast envelope. PEG caused severe destruction of the chloroplast envelope compared with NaCl, however thylakoids did not swell. Our observations suggested that in salt-treated rice plants, the ionic effects induced swelling of thylakoids and the osmotic effects caused the destruction of chloroplast envelope.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

等渗的盐分和水分胁迫对杠柳种子萌发的影响

通过等渗的NaCl和PEG溶液模拟盐分和水分胁迫,设置渗透梯度,在控制条件下对杠柳种子的萌发过程中总萌发率、幼苗鲜重、胚根生长、种子活力、发芽值等指标系统研究,对种子萌发率与渗透势之间关系进行回归分析,主要结果包括：（1）杠柳种子萌发过程中总萌发率、幼苗鲜重、活力指数和发芽值四项指标均随NaCl和PEG溶液的渗透势降低逐渐下降,综合研究活力指数和发芽值表明渗透势≥-0.5 MPa和≤-1.4 MPa时,PEG对种子萌发抑制作用大于NaCl,其他情况相反;（2）杠柳种子逐日萌发率和胚根日变化研究表明,与NaCl相比,PEG推迟杠柳种子萌发,并且对胚根增长抑制作用较大;（3）建立盐分和水分胁迫条件下种子萌发率与渗透势回归方程,发现杠柳种子在PEG胁迫下的萌发临界值和极限值为-1.0和-1.4 MPa,在NaCl胁迫下是-0.9和-1.3 MPa,解除胁迫条件,不同处理的杠柳种子复水萌发率均达到100%。说明杠柳具有良好的耐盐抗旱的特性。     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.