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Aim Sexually transmitted infections remain a neglected area of research in geriatrics. However, in the global aging societies, sexual health among the middle-aged and older adults is an emerging public concern. High-income countries are facing a resurgence of syphilis cases among young generations, but little is known about its prevalence in older populations. We aimed to investigate the national trend of syphilis cases in Japan. Methods This nationwide observational study used the publicly-available database (2009-2019) to calculate crude and age-adjusted incidence rates of syphilis per 100 000 population by age, sex and clinical stage. We collected data from patients aged >= 50 years and performed joinpoint regression analysis to estimate long-term trends and average annual percentage changes (AAPCs). Results The total number of patients with syphilis increased about 8-fold from 165 in 2009 to 1280 in 2019. AAPCs of crude incidence rates significantly increased in every age category; 33.2% in 50-59 years, 23.8% in 60-69 years and 20.9% in >= 70 years. Age-adjusted incidence rates have surged at AAPCs of 28.7% in men and 23.1% in women, reaching 4.09 in men and 0.71 in women in 2019. By clinical stage, marked increases were observed in primary (AAPCs, 42.3% in men and 41.6% in women) and secondary syphilis (AAPCs, 24.9% in men and 24.2% in women). Conclusions An up-toward trend of syphilis among people aged >= 50 years was observed. The importance of sexual health among older people should be highlighted in this aging Japanese society. Geriatr Gerontol Int 2022; center dot center dot: center dot center dot-center dot center dot.

Aim: An increasing number of older adults in Japan are at an increased risk of traffic accident. This study aimed to investigate the prevalence of potential underlying medical factors that increase the risk of motor vehicle accidents (MVAs) among older people. Methods: This cross-sectional observational study was conducted in 11 medical institutions in Japan using self-administered questionnaires and physical examination from January to May 2021. The background and social data, data on the use of nursing care insurance, and clinical data suggestive of polypharmacy, sarcopenia, cognitive impairment, and frailty/oral frailty were obtained. The prevalence of these factors was compared between everyday and occasional drivers. Results: Data of 127 patients were collected; their median (interquartile range) age was 73 (70–78) years. Of the total participants, 82 were men (64.6%) and 45 were women (35.4%). There were 77 everyday drivers and 50 occasional drivers. Of these, 121 (95.3%) had not applied for nursing care insurance, but the numbers of those who required help 1 and 2 were 1 (0.8%) and 3 (2.4%), respectively. Prevalence of medical factors was as follows: polypharmacy, 27.6%; sarcopenia, 8.7%; dementia, 16.4%; frailty, 15.0%; and oral frailty, 54.3%; it was not significantly different between every day and occasional drivers. Intention to return the car license was significantly higher among the occasional drivers (2.6% vs. 14.0%; odds ratio: 6.7, 95% confidence interval: 1.2–70.6, P=0.024). Conclusion: We uncovered the prevalence of medical factors that increase the risk of MVAs among older people aged ≥65 years in our community.

Netherton's syndrome, a rare congenital disorder, is clinically characterized by chronic dermatologic disorders such as ichthyosiform erythroderma and ichthyosis linearis circumflexa. Curable treatment is yet to be established, and corticosteroid ointment is required to maintain good dermatological condition. Because of the permanent skin barrier impairment, patients with Netherton's syndrome are considered to be vulnerable to cutaneous infections. However, its clinical characteristics are yet to be elucidated due to the limited number of reported cases. Herein, we describe the clinical course of a patient who developed persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. A 19-year-old Japanese woman who had been diagnosed with Netherton's syndrome in her infancy and had been applying topical corticosteroid agents all over her body since her then, was referred to our hospital because of persistent MRSA bacteremia and secondary adrenal insufficiency. The patient was diagnosed with a central line-associated bloodstream infection and was appropriately treated with antibiotics and corticosteroid therapies. We assume that the damaged skin barrier due to the congenital dermatological disorder causes a disruption in the normal bacterial flora of the skin, leading to the invasion of harmful bacteria, such as S. aureus. In addition, internal (humoral immunodeficiency by decreased antibody against bacterial polysaccharide antigens) and external (prolonged and systemic use of corticosteroid ointment) factors bring about an immunodeficiency state in such patients. We highlight that in the absence of radical treatment, clinicians need to recognize that patients with Netherton's syndrome are vulnerable to bacterial infections owing to the mixture of immunosuppressive factors.

To promote antimicrobial stewardship, we studied antimicrobial prescription rates for uncomplicated cystitis, a common outpatient disease requiring antibiotic treatment. This multicenter retrospective study was performed from January 1, 2018, to December 31, 2020, in Japan, targeting outpatients aged ≥ 20 years whose medical records revealed International Classification of Diseases (ICD-10) codes suggesting uncomplicated cystitis (N300). The data of 1445 patients were collected and that of 902 patients were analyzed. The overall median patient age was 71 years and a proportion of those aged less than 50 years was 18.8% with a female dominance (82.6%). Antimicrobials were prescribed for 884 patients (98.0%) and a total of 623 patients (69.1%) were treated with broad-spectrum drugs, including fluoroquinolones (36.0%), third-generation cephalosporins (29.9%) and faropenem (3.1%). A logistic regression model revealed that the broad-spectrum agents were significantly prescribed for the older patients, male patients, and those who visited internists. Recurrence was observed in 37 (4.1%) cases, and the multivariate analysis suggested any of age, sex, or antimicrobial types were not associated with the recurrence. Collectively, approximately two-thirds of antimicrobials prescribed for uncomplicated cystitis were broad-spectrum agents. The present data would be an indicator for antimicrobial prescriptions in uncomplicated cystitis in Japan.

Foliar uptake of nitrogen dioxide (NO(2)) is governed by its reactive absorption mechanism, by which NO(2) molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO(2) remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO(2), as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm (15)N-labeled NO(2) for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO(2) using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO(2) in leaves without MSX treatment was 587.0 nmol NO(2)/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO(2) by half. Thus, half of the foliar uptake of NO(2) is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO(2) is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.

Exposure of intact Arabidopsis leaves to 40 ppm nitrogen dioxide (NO2) in light resulted almost exclusively in nitration of PsbO1, PsbO2, and PsbP1 of photosystem II (PSII), with minor nitration of four non-PS II proteins, including peroxiredoxin II E, as reported previously. Our previous findings that light-triggered selective nitration of PsbO1 decreased oxygen evolution and that inhibition of photoelectric electron transport inhibited nitration of PsbO1 implied that the nitratable tyrosine residue of PsbO1 is redox-active. However, whether the nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active is unknown. In this study, we determined the oxygen evolution and maximal photochemical efficiency of PSII in intact Arabidopsis leaves following exposure to 40 ppm NO2 in light and found that these parameters were decreased to 60 and 70% of the non-exposed control, respectively. Because PsbO1, PsbO2, and PsbP1 accounted for > 80% of anti-3-nitrotyrosine antibody signal intensities, observed decreases in the oxygen evolution and maximal photochemical efficiency of PSII were mainly attributable to nitration of the tyrosine residues of these PSII proteins. Thus, it is postulated that nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active, as in the case of PsbO1. A new hypothetical model is proposed.

Treatment of isolated Arabidopsis thaliana thylakoid membranes with nitrogen dioxide (NO2) induces selective nitration of the tyrosine residue at the ninth amino acid (9Tyr) of PsbO1. This selective nitration is triggered by light and is inhibited by photosynthetic electron transport inhibitors. Therefore, we postulated that, similar to 161Tyr of D1 (YZ), 9Tyr of PsbO1 is redox active and is selectively oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical that is highly susceptible to nitration. This tyrosyl radical may combine rapidly at diffusion-controlled rates with NO2 to form 3-nitrotyrosine. If this postulation is correct, the nitration of 9Tyr of PsbO1 should decrease oxygen evolution activity. We investigated the effects of PsbO1 nitration on oxygen evolution from isolated thylakoid membranes, and found that nitration decreased oxygen evolution to ≥ 0% of the control. Oxygen evolution and nitration were significantly negatively correlated. This finding is consistent with redox active properties of the 9Tyr gene of PsbO1, and suggests that PsbO1 9Tyr acts as an electron relay, such as YZ in the photosystem II oxygenic electron transport chain.

It is known that when plant leaves are exposed to exogenously applied nitrogen dioxide (NO2), nitrogen derived from NO2 is reduced to amino acid nitrogen. However, whether this is the sole metaboli...

To elucidate the stimulation of leaf growth by atmospheric nitrogen dioxide (NO2), we performed a kinematic analysis of the eighth leaves of Arabidopsis thaliana (accession C24) plants grown for 17–35 days after sowing in the presence or absence of 50 ppb NO2 (designated +NO2 plants and –NO2 plants, respectively). We found that the peak and mean values of the relative rates of leaf expansion, cell division and cell expansion were always greater in +NO2 plants than in –NO2 plants. No evidence for prolonged duration was obtained. Thus, NO2 treatment increased the rates of both cell proliferation and enlargement to increase leaf size. Furthermore, a fold-change analysis showed that cell proliferation and enlargement differentially regulated NO2-induced leaf expansion.

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr36 of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the two treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ∼100-fold greater than that of nitrite.

In this communication, we review our work over two decades on air-pollutant-philic plants that can grow with air pollutants as the sole nutrient source. We believe that such plants are instrumental in mitigating air pollution. Our target air pollutant has been atmospheric nitrogen dioxide (NO2), and our work on this subject has consisted of three parts: Variation in plants’ abilities to mitigate air pollutants among naturally occurring plants, genetic improvement of plants’ abilities to mitigate air pollutants, and the plant vitalization effect of NO2. So far, an estimation of the half-life of nitrogen derived from NO2 uptake in plants belonging to the 217 taxa studied to date has shown no plants to be naturally occurring air-pollutant-philic. However, we found that an enormous difference exists in plants’ ability to uptake and assimilate atmospheric NO2. Future studies on the causes of this process may provide an important clue to aid the genetic production of plants that are effectively air-pollutant-philic. Both genetic engineering of the genes involved in the primary nitrate metabolism and genetic modification by ion-beam irradiation failed to make plants air-pollutant-philic, but mutants obtained in these studies will prove useful in revealing those genes critical in doing so. During our study on air-pollutant-philic plants, we unexpectedly discovered that prolonged exposure of plants to a sufficient level of NO2 activates the uptake and metabolism of nutrients that fuel plant growth and development. We named this phenomenon “the plant vitalization effect of NO2” (PVEON). Investigations into the mechanisms and genes involved in PVEON will provide an important clue to making plants air-pollutant-philic in the future.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO(2)) results in the selective nitration of specific proteins, such as PsbO1. The 9th tyrosine residue ((9)Tyr) of PsbO1 has been identified as the nitration site. This nitration is triggered by light and inhibited by photosynthetic electron transport inhibitors. During protein nitration, tyrosyl and NO(2) radicals are formed concurrently and combine rapidly to form 3-nitrotyrosine. A selective oxidation mechanism for (9)Tyr of PsbO1 is required. We postulated that, similar to (161)Tyr of D1, (9)Tyr of PsbO1 is selectively photo-oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical. In corroboration, after reappraising our oxygen evolution analysis, the nitration of PsbO1 proved responsible for decreased oxygen evolution from the thylakoid membranes. NO(2) is reportedly taken into cells as nitrous acid, which dissociates to form NO(2)(−). NO(2)(−) may be oxidized into NO(2) by the oxygen-evolving complex. Light may synchronize this reaction with tyrosyl radical formation. These findings suggest a novel role for PsbO1 in photosynthetic electron transport.

A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while HATS activity and transcripts for HvNRT2 and HvNAR2 were markedly repressed. An affinity column binding analysis using recombinant proteins suggests that the C-terminus of HvNRT2.1 is possibly involved in its binding to the HvNAR2.3 central region and that the Ser463 present in the HvNRT2.1 C-terminus plays a role in the binding ability.

Atmospheric nitrogen dioxide (NO(2)) is an environmental oxidant that is removed through direct uptake by foliage, but plant responses to this highly reactive gas are not well understood at the molecular level. From NO(2)-exposed leaves of a woody azalea (Rhododendron mucronatum), we cloned two cDNAs (RmGLP1 and RmGLP2) for germin-like proteins (GLPs), a group of ubiquitous plant proteins that have been implicated in various plant physiological and developmental processes. Quantitative analysis of mRNA expression, together with immunoblotting data, showed that foliar exposure to NO(2) caused a robust induction of these GLP-encoding genes. When produced in tobacco cell culture, recombinant RmGLP2 was secreted into the apoplast, where it exhibited superoxide dismutase activity. RmGLP1 and RmGLP2 represent the first examples of plant genes that are responsive to airborne NO(2). These enzymes might have a potential role in extracellular defense mechanisms through attenuation of interactions between reactive nitrogen and oxygen species.

As reported previously, atmospheric nitrogen dioxide (NO2) at an ambient level increased plant size and the contents of cell constituents. We investigated this effect of atmospheric NO2 on decontamination of cadmium (Cd) by kenaf (Hibiscus cannabinus). Seventeen-day-old seedlings of kenaf were grown in air either with NO2 or without NO2. (Plants were exposed to 100 +/- 50 ppb NO2 for 10 d under irrigation of 0.1% Hyponex supplemented with 20 microM CdCl2.) Plants were then harvested and the biomass of stems, leaves, and roots, as well as the content of Cd in the organs, was determined. The stem and root biomass per plant were 1.25-1.27-fold greater in +NO2 plants than in -NO2 plants. The Cd content per stem was more than 30% greater in +NO2 plants than in -NO2 plants.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.

In order to screen for the best species for mitigating nitrogen dioxide (NO2) by plants at urban levels, we investigated assimilation of nitrogen dioxide by 70 taxa of woody plants that are mostly utilized as roadside trees. They were fumigated with 15N-labeled NO2 at 0.1 microl l(-1) for 8h, and the amount of reduced nitrogen derived from NO2 (in mg Ng(-1) dry weight) in the leaves (designated NO2 assimilation capability hereafter) were determined. Data were analyzed in the comparison with the previously reported ones obtained at 4 microl l(-1) NO2. Among the 70 taxa, the value of NO2 assimilation capability differed by a factor of 122 between the highest (Prunus yedoensis; 0.061) and the lowest (Cryptomeria japonica; 0.0005). Based on the analysis of NO2 assimilation capability values at 0.1 and 4 micro l(-1) NO2, the 70 taxa of woody plants appeared to be classified into four types; those of high NO2 assimilation and high NO2 resistance, those of high NO2 assimilation but low NO2 resistance, those of low NO2 assimilation and low NO2 resistance, and those of low NO2 assimilation but high NO2 resistance. The first, second, third and fourth types include 13, 11, 35 and 11 taxa, respectively. The broad-leaf deciduous trees may have advantages of high biomass and fast growth as compared with woody plants of other habits. Thus, four broad-leaf deciduous species, Robinia pseudo-acacia, Sophora japonica, Populus nigra and Prunus lannesiana, were concluded here to be the best phytoremediators for the urban air.

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The ob­tained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indi­cates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

Shoot explants cut from aseptically grown Ficus thunbergii plants were irradiated with 12 C 5� , 12 C 6� and 4 He 2� ion beams of 10 to 200 Gray (Gy). After irradiation, the explants were allowed to regenerate. The regeneration frequency (RF), or percent explants bearing regenerated shoots to the total number of explants irradiated, decreased in a dose- dependent manner. Morphological changes in leaves and stems were observed in regenerants. The morphological change frequency (MF), or percent regenerants bearing morphologically changed shoots to the total number of regenerants peaked at 15 to 25% at the dose of each ion beam that gave an RF of about 80% (except for 4 He 2� ). A negative correlation was observed between the linear energy transfer (LET) and ion beam dose both in RF and MF. The present results strongly suggest that ion beam irradiation can induce mutations in the genome of F. thu nbergii.

A 507-bp nuclear matrix attachment region (MAR) sequence (designated TJ1) was flanked at both at 5� and 3� ends of the expression cassette for a modified green fluorescent protein (GFP) gene of the 35 W-sGFP(S65T) in four different orientations. A plasmid p507TH bearing a 507-bp non-MAR sequence from yeast at both ends of the cassette also was constructed. These five plasmids and 35 W-sGFP(S65T) were separately delivered into cultured tobacco BY-2 cells by particle bombardment. The yield of stable transformants that showed a uniform green fluorescence was determined. Southern blot analysis confirmed the integration of the GFP gene in tobacco genome. Transformant yields of the two TJ1- lacking plasmids were not significant. Transformant yields appeared to be increased by more than 2 times by the presence of TJ1-MAR sequences. The orientation of TJ1-MAR sequences did not influence the transformant yield.

We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin alpha5beta1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin alpha5beta1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of alpha5 and beta1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin- mediated manner.

The production of a novel "nitrogen-dioxide-philic plant" that can grow with NO 2 as the sole nitrogen source was investigated as a possible solution to pollution. 217 plant taxa were screened for the assimilation of NO 2 , among them 50 wild herbaceous plants collected from roadsides, 60 cultivated herbaceous plants and 107 cultivated woody plants. It was discovered that the NO 2 -derived reduced nitrogen [μg N/mg dry weight] differed by a factor of 657 between the highest (Eucalyptus viminalis, 6.57) and lowest (Tillandsia ionantha and T. caputmedusae, 0.01) level in the 217 taxa. The capability of plants to assimilate NO 2 was also found to change in a family-dependent manner. Compositae, Myrtaceae, Salicaceae and Solanaceae had taxa with high NO 2 assimilation capability, while none of the Gramineae, Theaceae and Rosaceae plants examined showed high NO 2 assimilation capability. The 217 taxa included two species of the Solanaceae family (tobacco and petunia), both of which showed a high NO 2 assimilation and their reduced nitrogen derived from NO 2 was the third and twenty-sixth highest. This suggested that the Solanaceae family includes plants of high NO 2 assimilation. Therefore, in this study eight taxa of the Solanaceae family and four taxa of other families were investigated for NO 2 assimilation in the same manner as reported previously by the fumigation of plants with 1 5 N-labelled NO 2 followed by the mass spectroscopic analysis based upon the reduced nitrogen derived from NO 2 . The eight taxa Solanaceae included tobacco (Nicotiana tabacum) and petunia (Petunia X hybrida), ground cherry (Physalis alkekengi var. franchetii), tomato (Lycopersicon esculentum), datura (Datura innoxia), potato vine (Solanum jasminoides), red pepper (Capsicum annuum) and green pepper (Capsicum annuum var. grossum SENDTN.). The four taxa included two ivy cultivars (Hedera helix "Pittsburgh" and Hedera helix "Wonder"), an evergreen creeper (Ficus thunbergii) and kenaf (Hibiscus cannabinus). Among the twelve taxa, kenaf, tobacco and ground cherry showed the highest, second and third highest values, respectively, of reduced nitrogen derived from NO 2 in plants, which reflects the capability to assimilate NO 2 -nitrogen. The quantitative significance of these findings is discussed in relation to the reduction of nitrogenous pollution by the atmospheric NO 2 pollution.

Summary The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.

When hypocotyl sections of Raphiolepis umbellata L. were cultured in a woody plant medium containing 8.9μM benzylaminopurine and 1.1μM α-naphthaleneacetic, adventitous shoots were formed. Fully developed plants were obtained in 3 months of the culture. A chimeric plasmid pENiR harboring cDNA of Arabidopsis thaliana nitrite reductase gene (nii) under the control of an enhanced promoter consisting of sequences from both cauliflower mosaic virus 35S promoter and tobacco mosaic virus omega regions and a nopaline synthase terminator was constructed and introduced to the hypocotyl sections by particle bombardment. A plasmid pCH bearing hygromycin phosphotransferase gene (hph) was cointroduced. Transformed shoots were selected for hygromycin resistance. Polymerase chain reaction analysis showed the presence of hph gene in 16 out of 2572 bombarded hypocotyl sections, and two of which were found to bear Arabidopsis nii gene. Translation of this gene in transgenic plants was confirmed by Western blot analysis.

In order to select individuals of Rhododendron mucronatum plants that have a high capacity to decontaminate nitrogen dioxide (NO 2 ), which is a major air pollutant, a number of lines prepared by cutting propagation were analyzed for their uptake of NO 2 . In 1996, 1452 different lines (referred to as the 96-plants) were fumigated with 4 μl/l 15 NO 2 for 8 hours, and the total nitrogen content of the leaves, derived from NO 2 , was determined and reflects the plant's NO 2 uptake capacity. A 56-fold difference was observed between the individuals with the highest and lowest capacities. About 100 lines each from the high-, middle- and low-capacity groups were propagated and assayed for NO 2 uptake in the following year (the 97-plants). The maximum variation in NO 2 uptake was 5.6 times. In 1998, 227 lines propagated from the 96-plants and 95 lines from the 97-plants were assayed for NO 2 uptake (the 98-plants). The variations in NO 2 uptake in 97- and 98-plants were a maximum of 4.9 and 5.2 times, respectively. The reason(s) for the disappearance of the initial large variation in NO 2 uptake is (are) not yet known. Those lines showing high and low NO 2 uptake capacities will be useful when studying the genes involved in this important roadside tree.

DNA sequences of transgenic loci in the transgenes of tobacco and Arabidopsis were analyzed. Almost all transgenic loci studied contained cis elements characteristic of nuclear matrix attachment regions (MARs). A MAR sequence isolated from one such locus of tobacco cells was cloned into the original transformation vector, and introduced into tobacco cells. The presence of the MAR sequence resulted in a five- to tenfold increase in the transformant yields, and it appeared to stimulate both the expression of the transgene and its integration into the host genome. A search using the BLAST program revealed that the transgenic loci contained junction region sequences that may have originated from different chromosomes of the nuclear genome and from the chloroplast genome.

Prolonged exposure of plants to a sufficient level of atmospheric nitrogen dioxide (NO2) activates the uptake and metabolism of nutrients that fuel plant growth and development, a phenomenon termed the plant vitalization effect of NO2. This study examined the effect of NO2 on the fruit yield of tomato (Solanum lycopersicum L. cv. Micro-Tom). Double exposure chambers, designated ±NO2 chambers, were placed in a confined greenhouse. The air entering the −NO2 chamber was scrubbed of nitrogen oxides including NO2, and the air entering the +NO2 chamber was supplemented with NO2 (50 ± 10 ppb). Two-week-old tomato seedlings that had been grown in the −NO2 chamber after sowing were then grown in a +NO2 or −NO2 chamber for the remainder of the growth period until 96 days after sowing. Growth in the +NO2 chamber led to a significant increase in fruit yield of approximately 40% as compared to growth in the −NO2 chamber. This increase in fruit yield was accompanied by acceleration of flowering time by 3.2 days and an increase in flower number per plant of up to 60%. These results show that exposure to NO2 increases fruit yield via stimulation of flowering in tomato.

Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach + Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO2 assimilation by analysis of the reduced nitrogen derived from NO2(NO2-RN). Twelve independent transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between the total NiR mRNA and total NiR protein contents (r = 0.74), between the total NiR protein and NiRA (r = 0.71), and between NiRA and NO2-RN (r = 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P < 0.01), and three had significantly higher NO2-RN (P < 0.01). Each of the latter three had one to two copies of spinach NiR cDNA per haploid genome. The NiR flux control coefficient for NO2 assimilation was estimated to be about 0.4. A similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR is a controlling enzyme in NO2assimilation by plants.

Summary The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3–, NO2– or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3–-containing medium. N2O emission from clone 271 grown on NO3–-containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg–1 incorporated N week–1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2–-containing medium and its emission rate increased with increasing NO2– levels in plants. We speculate that NO3– is reduced to NO2– and that a part of NO2– is metabolized to N2O in clone 271.