Modification of soy protein isolate by Maillard reaction and its application in microencapsulation of Limosilactobacillusreuteri.

Limosilactobacillusreuteri was encapsulated using Maillard-reaction-products (MRPs) of soy protein isolate (SPI) and α-lactose monohydrate by freeze-drying. The mixed solution of SPI and α-lactose monohydrate was placed in a water bath at 89°C for 160 min for Maillard reaction, and then freeze-dried to obtain MRPs. The effects of Maillard reaction on functional characteristics of MRPs and the properties of MRPs-microcapsules were studied. SDS-PAGE indicated that SPI subunit reacted with lactose to form a polymer, and the band of MRPs disappeared around the molecular weights of 33, 40, 63, and 100 kDa. Compared with SPI, the emulsion stability, emulsion activity, foaming capacity, foam stability, and gel strength of MRPs were increased by 259%, 55.71%, 82.32%, 58.53%, and 3266%, respectively. The results of Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and scanning electron micrographs confirmed that the protein structure also changed significantly. Then, MRPs were used as wall material to prepare L. reuteri microcapsules. Physical properties and viable counts of L. reuteri during the simulated gastrointestinal digestion and storage period were determined. The particle size of MRPs-microcapsules (68 µm) was smaller than that of SPI-microcapsules (91 µm). The viable counts of L. reuteri in simulated gastrointestinal digestion and after storage for 30 days were improved. The modifications with Maillard reaction can improve emulsification, foaming, and gel strength of SPI, and MRPs could be used as a new type of wall material in the production of L. reuteri microcapsules.

Inhibition of miR-1298-5p attenuates sepsis lung injury by targeting SOCS6.

Sepsis is one of the leading causes of morbidity and mortality and a major cause of acute lung injury (ALI). carried by exosomes play a role in a variety of diseases. However,there are not many studies of exosomal miRNAs in sepsis and sepsis lung injury.miR-1298-5p and suppressor of cytokine signaling 6 (SOCS6) were silenced or overexpressed in human bronchial epithelial cells (BEAS-2B). PKH-67 Dye was used to trace exosome endocytosis. Cell permeability was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC dextran flux. ELISA kits were used for cytokine detection. Quantitative RT-PCR and western blots were used to evaluate gene expression. miR-1298-5p was elevated in exosomes from patients with sepsis lung injury (Sepsis_exo). Treatment of BEAS-2B cells using Sepsis_exo significantly inhibited cell proliferation, and induced cell permeability and inflammatory response. miR-1298-5p directly targeted SOCS6. Overexpressing SOCS6 reversed miR-1298-5p-induced cell permeability and inflammatory response. Inhibition of STAT3 blocked SOCS6-silencing caused significant increase of cell permeability and inflammation. Exosomes isolated from patients of sepsis lung injury increased cell permeability and inflammatory response in BEAS-2B cells through exosomal miR-1298-5p which targeted SOCS6 via STAT3 pathway. The findings highlight the importance of miR-1298-5p/SOCS6/STAT3 axis in sepsis lung injury and provide new insights into therapeutic strategies for sepsis lung injury.

Kinetic changes in virology, specific antibody response and imaging during the clinical course of COVID-19: a descriptive study.

BACKGROUND: To explore the kinetic changes in virology, specific antibody response and imaging during the clinical course of COVID-19. METHODS: This observational study enrolled 20 patients with COVID-19, who were hospitalized between January 20-April 6, 2020, in the two COVID-19 designated hospitals of Zhoushan, Zhejiang and Rushan, Shandong, China, The laboratory findings, imaging, serum response to viral infection, and viral RNA level in the throat and stool samples were assessed from onset to recovery phase in patients with COVID-19. RESULTS: SARS-COV-2 RNA was positive as early as day four. It remained positive until day 55 post-onset in the sputum-throat swabs and became negative in most cases (55%) within 14 days after onset. Lymphocytopenia occurred in 40% (8/20) of patients during the peak infection period and returned to normal at week five. The most severe inflammation in the lungs appeared in week 2 or 3 after onset, and this was completely absorbed between week 6 and 8 in 85.7% of patients. All patients had detectable antibodies to the receptor binding domain (RBD), and 95% of these patients had IgG to viral N proteins. The antibody titer peaked at week four. Anti-S IgM was positive in 7 of 20 patients after week three. CONCLUSIONS: All COVID-19 patients in this study were self-limiting and recovered well though it may take as long as 6-8 weeks. Our findings on the kinetic changes in imaging, serum response to viral infection and viral RNA level may help understand pathogenesis and define clinical course of COVID-19.

LPS-Treated Podocytes Polarize Naive CD4+ T Cells into Th17 and Treg Cells.

AIM: Our study is aimed at investigating whether Lipopolysaccharide- (LPS-) treated podocytes could polarize naive CD4+ T cells into different subsets in vitro. MATERIALS AND METHODS: Podocytes and mouse bone marrow-derived dendritic cells (BMDCs) were first cultured with 25 µg/ml LPS for 6 hours, respectively. Then, naive CD4+ T cells were cocultured with the LPS-treated podocytes or BMDCs at a ratio of 1 : 1 or 1 : 1 : 1. After 48 hours, we collected the suspended cells and supernatant from all groups to measure T helper (Th)17 cells, regulatory T (Treg) cells, and cytokine concentration. RESULTS: We observed the expression of CD80 and major histocompatibility complex class II molecule (MHC II) in podocytes but did not found the upregulation of them after treating podocytes with LPS. LPS-treated podocytes could induce naive CD4+ T cells to Th17 cells and Treg cells with a higher ratio of Th17/Treg than BMDCs. Possible interaction between podocytes and BMDCs may exist in the induction process of Th17 cells and Treg cells. CONCLUSION: Our study proved that CD80 and MHC II were constitutively expressed in podocytes but not upregulated by LPS. LPS-treated podocytes could polarize naive CD4+ T cells into Th17 and Treg cells and affect the Th17/Treg balance and may incline to cause a Th17 response.

Management of venous perforation during central venous catheterization in hemodialysis patients: Three case reports.

RATIONALE: In recent years, central venous catheterization (CVC) has become widely used for hemodialysis patients. Based on 3 cases, we discussed the detection and management of venous perforation at an early stage. PATIENTS CONCERNS: Patients 1 (male, 77 years), 2 (male, 82 years), and 3 (male, 30 years) were diagnosed with uremia and underwent hemodialysis. DIAGNOSES: Computed tomography suggested pneumomediastinum in patient 1 and pneumothorax in patient 2 after a replacement of the temporary hemodialysis catheter. In patient 3, X-ray suggested that the tip of the catheter was approximately at the plane of the fifth thoracic vertebrate after the temporary catheter was placed. INTERVENTIONS: In patients 1 and 2, the catheters were maintained where they were for about 2 weeks until a false lumen formed outside the catheter. In patient 3, the catheter was withdrawn at once when vein perforation was observed. OUTCOME: In patients 1 and 2, the catheters were adjusted successfully under digital subtraction angiography (DSA) guidance 2 weeks later. In patient 3, hemothorax developed, and a total of approximately 1000 mL of bloody fluid was drained. LESSONS: When venous perforation occurs during CVC, it is safer and more reliable to adjust or withdraw the catheter under DSA guidance after a false lumen forms outside the catheter.

Chemical conversion of human lung fibroblasts into neuronal cells.

It has been previously reported that human embryonic fibroblasts and mouse embryonic fibroblasts can be converted into neuronal cells using chemical agents, along with forced expression specific transcriptional factors. However, the materials required for reprogramming in these approaches presents major technical difficulties and safety concerns. The current study investigated whether a cocktail of small molecules can convert human lung fibroblast cells into neurons. The small molecules valproic acid, CHIR99021, DMH1, Repsox, forskolin, Y­27632 and SP600125 (VCHRFYS) were used to induce MRC­5 cells into neuronal cells in vitro. Neuronal markers were analyzed by immunofluorescence staining. The gene profiles were analyzed by reverse transcription­quantitative polymerase chain reaction. MRC­5 is a human lung fibroblast cell line derived from normal lung tissue of a 14­week­old male fetus. The results of the current study demonstrated that MRC­5 fibroblasts can be directly converted into neuronal cells using a cocktail of seven small molecules (VCHRFYS), with a yield of ~90% Tuj1­positive cells after 7 days of induction. Following a further maturation period, these chemically-induced neurons possessed neuronal morphology and expressed multiple neuron­specific genes. In conclusion, a cocktail of small molecules that can convert fibroblasts MRC­5 cells into functional neurons without the exogenous genetic factors was identified, which has the potential to be useful in neurological disease therapy.

[Intermittent convulsions for 1.5 years and psychomotor retardation in a girl].

The study reports a girl with pyridoxine-dependent epilepsy. The girl was admitted at the age of 2 years because of intermittent convulsions for 1.5 years and psychomotor retardation. She had a history of "hypoxia" in the neonatal period. At the age of 5 months recurrent epileptic seizures occurred. The child was resistant to antiepileptic drugs, and had many more seizures when she got cold or fever. She also had a lot of convulsive status epilepticus. No discharges were found during several video-EEG monitorings. Cerebral MRI examinations showed normal results. So Dravet syndrome was clinically suspected. ALDH7N1 gene mutation analysis revealed two heterozygote mutations, and pyridoxine-dependent epilepsy was thus confirmed. Seizures were generally controlled after pyridoxine supplementation.

Clinical characteristics and advantages of primary peripheral micro-sized lung adenocarcinoma over small-sized lung adenocarcinoma.

OBJECTIVES: Micro-sized lung adenocarcinoma with a tumour of 1.0 cm or less could help identify the patients who would undergo the surgery treatment with limited resection; however, its clinical characteristics and survival rates remain unclear and are to be tested further. METHODS: Histology, lymphatic metastasis, surgical procedure and survival rates of 366 lung adenocarcinoma patients (from January 2007 to December 2013) with a tumour of 2.0 cm or less were analysed retrospectively. Among these patients, 175 had a primary tumour with a diameter of 1.0 cm or less and 191 had a tumour of 1.1-2.0 cm. The survival of 366 patients was evaluated by the restricted mean survival time (RMST) test, and the risk factors were assessed by multivariable analysis. RESULTS: Larger lesion had a significant relation to old age, male sex, preoperatively carcinoembryonic antigen (CEA) positive, invasive adenocarcinoma (IAC) and advanced-stage disease (P < 0.0001, P = 0.001, P = 0.001, P < 0.0001 and P < 0.0001, respectively). Patients with adenocarcinoma in situ (AIS)/minimally invasive adenocarcinoma obtained a better prognosis than those with IAC (5-year overall survival rate: 98.5 vs 84.3%, P = 0.001; disease-related survival rate: 98.5 versus 85.2%, P = 0.001). The 5-year overall survival rates of patients with a tumour less than 1.0 cm in comparison with 1.1-2.0 cm were 100.0 and 88.4% (P < 0.001), whereas the 5-year lung adenocarcinoma-specific survival rates were 100.0 and 89.0% (P < 0.001), respectively. Multivariable analysis for prognosis of lung adenocarcinoma patients with a tumour 2.0 cm or less in diameter revealed that histology, lymphatic metastasis and advanced pathological stage affected the 5-year overall and disease-related survival rates unfavourably (P < 0.0001, 0.002, 0.001; and P < 0.0001, 0.005, 0.001, respectively), whereas tumour size did not have an obvious influence on survival. CONCLUSIONS: Micro-sized lung adenocarcinoma (1.0 cm or less) had specific clinical characteristics and more favourable survival rates. These tumours and a subtype of AIS evaluated by computed tomography images or intraoperative frozen section may be appropriate candidates for a limited resection without mediastinal lymph node dissection.

Evidence for transcriptional interference in a dual-luciferase reporter system.

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

Tim-4 Inhibits NO Generation by Murine Macrophages.

OBJECTIVE: T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation. METHODS: Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot. RESULTS: Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages. CONCLUSION: These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.

Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies.

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin. METHODS: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions. RESULTS: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site. MAJOR CONCLUSION: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model. GENERAL SIGNIFICANCE: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.

Tim-3 expression by peripheral natural killer cells and natural killer T cells increases in patients with lung cancer--reduction after surgical resection.

BACKGROUND: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+ natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. MATERIALS AND METHODS: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim- 3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controls by flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patient cohort. RESULTS: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3- CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 and p=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevated Tim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages (III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression was significantly reduced on after surgical resection of the primary tumor (p<0.01). CONCLUSIONS: Tim-3 expression in natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lung cancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.

Differential expression of miR-125a-5p and let-7e predicts the progression and prognosis of non-small cell lung cancer.

Aberrant expression of various microRNAs (miRNA) has shown diagnostic and prognostic significance in non-small cell lung cancer (NSCLC). qRT-PCR analysis confirmed altered expression of miR-125a-5p, let-7e, miR-30a, miR-30e and miR-30e-3p in 70 paired tissue and serum samples from NSCLC patients. The reduced expression of miR-125a-5p, let-7e and miR-30e was strongly associated with NSCLC dedifferentiation. The lost expression of miR-125a-5p and let-7e was associated with shorter overall survival and let-7e was an independent prognostic factor for NSCLC patients. These five miRNA expressions should be further evaluated as biomarkers for the early detection and prognosis of NSCLC patients.

Reactions of amino acids, peptides, and proteins with oxidized metabolites of tris(p-carboxyltetrathiaaryl)methyl radical EPR probes.

Oxidation of the tris(p-carboxyltetrathiaaryl)methyl (TAM) EPR radical probe, TAMa(•), by rat liver microsomes (RLM) + NADPH, or horseradish peroxidase (HRP) + H2O2, or K2IrCl6, led to an intermediate cation, TAMa(+), which was treated with glutathione (GSH), with formation of an adduct, TAMa-SG(•), resulting from the substitution of a TAMa(•) carboxylate group with the SG group. L-α-Amino acids containing a strong nucleophilic residue (NuH), such as L-cysteine or L-histidine, also reacted with TAMa(+), with formation of radical adducts TAMa-Nu(•) in which a carboxylate group of TAMa(•) was replaced with Nu. Other less nucleophilic L-α-amino acids, such as L-arginine, L-serine, L-threonine, L-tyrosine, or L-aspartate, as well as the tetrapeptide H-(Gly)4-OH, reacted with TAMa(+) via their α-NH2 group, with formation of an iminoquinone methide, IQMa, deriving from an oxidative decarboxylation and amination of TAMa(•). Upon reaction of TAMa(+) with L-proline and L-lysine, N-substituted iminoquinone methide adducts, IQMa-Pro and IQMa-Lys, were formed. Finally, preliminary results showed that oxidation of TAMa(•) in the presence of bovine serum albumin (BSA), led to the covalent binding of TAMa-derived metabolites to BSA. Oxidation of another frequently used TAM probe, TAMb(•) (Oxo63), in the presence of GSH, N-acetyl-cysteine methyl ester, or histidine also led to TAMb-Nu(•) adducts equivalent to the corresponding TAMa-Nu(•) adducts, suggesting that the oxidative metabolism of such TAM(•) probes could lead to protein covalent binding. Moreover, the above data describe an easy access to new TAM radical EPR probes coupled to amino acids, peptides or proteins that could be useful for addressing various biological targets.

Toward stable electron paramagnetic resonance oximetry probes: synthesis, characterization, and metabolic evaluation of new ester derivatives of a tris-(para-carboxyltetrathiaaryl)methyl (TAM) radical.

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals, such as 1a ("Finland" radical), are useful EPR probes for oximetry. However, they are rapidly metabolized by liver microsomes in the presence of NADPH, with the formation of diamagnetic quinone-methide metabolites resulting from an oxidative decarboxylation of one of their carboxylate substituents. In an effort to obtain TAM derivatives potentially more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were completely characterized by UV-vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry. Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of dioxygen. These anions were much more stable than that of 1a and could be characterized by UV-vis spectroscopy, MS, and at the level of their protonated product. However, they were oxidized by O2, giving back to the starting ester radicals and catalyzing a futile cycle of O2 reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.

First combined in vivo X-ray tomography and high-resolution molecular electron paramagnetic resonance (EPR) imaging of the mouse knee joint taking into account the disappearance kinetics of the EPR probe.

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 µm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

Role of arginine guanidinium moiety in nitric-oxide synthase mechanism of oxygen activation.

Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate L-Arg first to N(omega)-hydroxyl-L-arginine and then to L-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the L-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of L-Arg analogues exhibiting a wide range of guanidinium pK(a) values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pK(a) value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.

Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (Cucumis sativus L.).

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.