Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)‐near infrared (NIR)‐I light imaging. If the skull optical clearing window is available for NIR‐II, the imaging depth will be further enhanced. Herein, we developed a vis‐NIR‐II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR‐II. Using a NIR‐II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis‐NIR‐II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR‐II laser‐induced targeted injury of cortical single vessel. This work enhances the ability of NIR‐II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.     

Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.     

Two Types of Clathrin-Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.     

Screening and identification of LMNB1 and DLGAP5, two key biomarkers in gliomas

Glioma is the most common primary cancer in the central nervous system. Despite advances in surgery, radiotherapy and chemotherapy over the past decades, the prognosis of glioblastoma patients remains poor. We aim to identify robust gene signatures to better understand the complex molecular mechanisms and to discover potential novel molecular biomarkers for glioma. By exploring {"type":"entrez-geo","attrs":{"text":"GSE16011","term_id":"16011"}}GSE16011, {"type":"entrez-geo","attrs":{"text":"GSE4290","term_id":"4290"}}GSE4290 and {"type":"entrez-geo","attrs":{"text":"GSE50161","term_id":"50161"}}GSE50161 data in Gene Expression Omnibus (GEO) database, we screened out 380 differentially expressed genes between non-tumor and glioma tissues, and further selected 30 hub genes through the Molecular Complex Detection (MCODE) plug-in in Cytoscape. In addition, LMNB1 and DLGAP5 were selected for further analyses due to their high expression in gliomas and were verified by using our cohort. Our study confirmed that LMNB1 and DLGAP5 were up-regulated in gliomas, and patients with high expression of LMNB1 or DLGAP5 had poor survival rate. Furthermore, silence of LMNB1 and DLGAP5 inhibited the proliferation of glioma cells. Together, LMNB1 and DLGAP5 were two potentially novel molecular biomarkers for diagnosis and prognosis of glioma.     

In vivo expansion of naïve CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration

Regulatory T cells (T_reg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T_reg cells was established. In IL-2 treated cancer patients a further T_reg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T_reg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T_reg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T_reg cells indicate IL-2 dependent thymic generation of naïve T_reg cells as a mechanism leading to increased frequencies of T_reg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T_reg cells after IL-2 administration. These results point to a more complex regulation of T_reg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T_reg cells.     

Neurovascular signals in amyotrophic lateral sclerosis

The image recapitulates the interplay between neuronal and vascular systems by highlighting the cellular players involved in the molecular signalling in the context of Amyotrophic Lateral Sclerosis.

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The neurovascular system (NVS) is a complex anatomic-functional unit that synergically works to maintain organs/tissues homeostasis of the entire body. NVS alterations have recently emerged as a common distinct feature in the pathogenesis of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Despite their undeniable involvement, neurovascular signalling pathways remain still far unknown in ALS. This review underlines the importance of endothelial, mural, and fibroblast cells as novel targets for ALS investigation and identifies in the interplay between neuronal and vascular systems the way to disclose novel molecular mechanisms behind the pathogenesis of ALS.     

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications.     

Tau regulates the localization and function of End‐binding proteins 1 and 3 in developing neuronal cells

The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU?/? neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation.

     

Neuronal influences are necessary to produce mitochondrial co‐localization with glutamate transporters in astrocytes

Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT‐1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co‐localization of GLT‐1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5‐tagged GLT‐1, pDsRed1‐Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co‐localization was quantified using Volocity software. Image analysis of confocal z‐stacks revealed no co‐localization between mitochondria and GLT‐1 in pure astrocyte cultures. Co‐culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT‐1. This co‐localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K⁺. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT‐1/mitochondrial co‐localization, however, in co‐cultures, Y27632 abolished mitochondrial:GLT‐1 co‐localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT‐1 distribution or GLT‐1: mitochondrial co‐localization. We conclude that the association between GLT‐1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.

     

Gartanin induces cell cycle arrest and autophagy and suppresses migration involving PI3K/Akt/mTOR and MAPK signalling pathway in human glioma cells

In central nervous system, glioma is the most common primary brain tumour. The diffuse migration and rapid proliferation are main obstacles for successful treatment. Gartanin, a natural xanthone of mangosteen, suppressed proliferation, migration and colony formation in a time‐ and concentration‐dependent manner in T98G glioma cells but not in mouse normal neuronal HT22 cells. Gartanin, at low micromole, led to cell cycle arrest in G1 phase accompanied by inhibited expression level of G1 cell cycle regulatory proteins cyclin D1, while increased expression level of cyclin‐dependent kinase inhibitor p27Kip1. In addition, the secretion and activity of matrix metalloproteinases 2/9 (MMP‐2/‐9) were significantly suppressed in T98G cells treated with gartanin, and it might result from modulating mitogen‐activated protein kinases (MAPK) signalling pathway in T98G glioma cells. Moreover, gartanin significantly induced autophagy in T98G cells and increased GFP‐LC3 punctate fluorescence accompanied by the increased expression level of Beclin 1 and LC3‐II, while suppressed expression level of p62. Gartanin treatment resulted in obvious inhibition of PI3K/Akt/mTOR signalling pathway, which is important in modulating autophagy. Notably, gartanin‐mediated anti‐viability was significantly abrogated by autophagy inhibitors including 3‐methyladenine (3‐MA) and chloroquine (CQ). These results indicate that anti‐proliferation effect of gartanin in T98G cells is most likely via cell cycle arrest modulated by autophagy, which is regulated by PI3K/Akt/mTOR signalling pathway, while anti‐migration effect is most likely via suppression of MMP‐2/‐9 activity which is involved in MAPK signalling pathway.     

Circular RNA circRNA_15698 aggravates the extracellular matrix of diabetic nephropathy mesangial cells via miR-185/TGF-β1

Circular RNAs (circRNAs) are a novel type of noncoding RNAs that modulate the pathogenesis of multiple diseases. Nevertheless, the role of circRNAs in diabetic nephropathy (DN) pathogenesis is still ambiguous. In the current study, our team aims to investigate the expression profiles of circRNAs in DN and identify the function of circRNA on mesangial cells. CircRNAs microarray analysis revealed dysregulated circRNA in db/db DN mice, and circRNA_15698 was validated to be upregulated in both db/db mice and mouse mesangial cells (SV40-MES13) that were exposed to high glucose (25 mM) using real-time polymerase chain reaction. Loss-of-functional experiments showed that circRNA_15698 knockdown significantly inhibited the expression levels of collagen type I (Col. I), collagen type IV (Col. IV), and fibronectin. Moreover, the cellular localization of circRNA_15698 was mainly in the cytoplasm. Bioinformatics tools and luciferase reporter assay confirmed that circRNA_15698 acted as a ‘sponge’ of miR-185, and then positively regulated the transforming growth factor-β1 (TGF-β1) protein expression, suggesting a circRNA_15698/miR-185/TGF-β1 pathway. Further validation experiments validated that circRNA_15698/miR-185/TGF-β1 promoted extracellular matrix (ECM)-related protein synthesis. In summary, our study preliminarily investigates the role of circRNAs in mesangial cells and ECM accumulation, providing a novel insight for DN pathogenesis.     

Fatty acid biosynthesis from glutamate and glutamine is specifically induced in neuronal cells under hypoxia

Hypoxia is involved in many neuronal and non‐neuronal diseases, and defining the mechanisms for tissue adaptation to hypoxia is critical for the understanding and treatment of these diseases. One mechanism for tissue adaptation to hypoxia is increased glutamine and/or glutamate (Gln/Glu) utilization. To address this mechanism, we determined incorporation of Gln/Glu and other lipogenic substrates into lipids and fatty acids in both primary neurons and a neuronal cell line under normoxic and hypoxic conditions and compared this to non‐neuronal primary cells and non‐neuronal cell lines. Incorporation of Gln/Glu into total lipids was dramatically and specifically increased under hypoxia in neuronal cells including both primary (2.0‐ and 3.0‐fold for Gln and Glu, respectively) and immortalized cultures (3.5‐ and 8.0‐fold for Gln and Glu, respectively), and 90% to 97% of this increase was accounted for by incorporation into fatty acids (FA) depending upon substrate and cell type. All other non‐neuronal cells tested demonstrated decreased or unchanged FA synthesis from Gln/Glu under hypoxia. Consistent with these data, total FA mass was also increased in neuronal cells under hypoxia that was mainly accounted for by the increase in saturated and monounsaturated FA with carbon length from 14 to 24. Incorporation of FA synthesized from Gln/Glu was increased in all major lipid classes including cholesteryl esters, triacylglycerols, diacylglycerols, free FA, and phospholipids, with the highest rate of incorporation into triacylglycerols. These results indicate that increased FA biosynthesis from Gln/Glu followed by esterification may be a neuronal specific pathway for adaptation to hypoxia.

     

Neuronal types and their percent distribution within the magnocellular nuclei of the human basal forebrain

Three neuronal types constituting the magnocellular nuclei of the human basal forebrain have been differentiated with the aid of preparations stained for both Nissl material and pigment deposits: type I = large multipolar neurons contain loosely packed and faintly stained lipofuscin granules occupying a large portion of the cell body; type II = large spindle-shaped neurons reveal a densely packed accumulation of coarse and intensely stained lipofuscin granules, and type III = small nerve cells, scattered among these large neuronal components, with only a small number of faintly stained lipofuscin granules. The determination of the projection areas of the somata of the three neuronal types has led to a distribution pattern with three peaks. The ratio of the nerve cell types has been evaluated: 73.6% type I; 8.6% type II, and 17.8% type III neurons.     

Real‐time imaging of photodynamic action in bacteria

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly‐sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single‐cell level. Imaging experiments in deuterated buffer strongly support a Type‐I mechanism for methylene blue and a very minor role of singlet oxygen.

     

Doublecortin induces mitotic microtubule catastrophe and inhibits glioma cell invasion

Doublecortin (DCX) is a microtubule (MT) binding protein that induces growth arrest at the G2–M phase of cell cycle in glioma and suppresses tumor xenograft in immunocompromised hosts. DCX expression was found in neuronal cells, but lacking in glioma cells. We tested the hypothesis that DCX inhibits glioma U87 cell mitosis and invasion. Our data showed that DCX synthesizing U87 cells underwent mitotic MT spindle catastrophe in a neurabin II dependent pathway. Synthesis of both DCX and neurabin II were required to induce apoptosis in U87 and human embryonic kidney 293T cells. In DCX expressing U87 cells, association of phosphorylated DCX with protein phosphatase-1 (PP1) in the cytosol disrupted the interaction between kinesin-13 and PP1 in the nucleus and yielded spontaneously active kinesin-13. The activated kinesin-13 caused mitotic MT catastrophe in spindle checkpoint. Phosphorylated-DCX induced depolymerization of actin filaments in U87 cells, down-regulated matrix metalloproteinases-2 and -9, and inhibited glioma U87 cell invasion in a neurabin II dependent pathway. Thus, localization of the DCX–neurabin II–PP1 complex in the cytosol of U87 tumor cells inhibited PP1 phosphatase activities leading to anti-glioma effects via (1) mitotic MT spindle catastrophe that blocks mitosis and (2) depolymerization of actin that inhibits glioma cell invasion.     

Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation

We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca²⁺/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser⁷⁴¹ to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, resulting in 60–70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser⁷⁴¹ in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS.     

Association of BDNF gene polymorphism with bipolar disorders in Han Chinese population

Recent data suggest that brain‐derived neurotrophic factor (BDNF) plays an essential role in neuronal plasticity and etiology of bipolar disorders (BPD). However, results from different studies have been inconsistent. In present study, 342 patients who met DSM‐IV (Diagnostic and Statistical Manual of Mental Disorders, 4th Edition) criteria for bipolar disorders type I (BPD‐I) or type II (BPD‐II) and 386 matched health controls were enrolled, and TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) were applied to detect the functional polymorphism rs6265 (Val66Met) of BDNF gene. Treatment response to lithium and valproate was retrospectively determined. The association between Val66Met polymorphism and BPD, treatment response to mood stabilizers, was estimated. The genotype and allele distribution of Val66Met polymorphism between BPD patients and control subjects showed significant difference (genotype: χ² = 6.18, df = 2, P = 0.046; allele: χ² = 5.01, df = 1, P = 0.025) with Met allele as risk factor for disease susceptibility (OR = 0.79, 95%CI as 0.64–0.97). The post hoc analysis interestingly showed that Met allele had opposite effect on the treatment response for BPD‐I and BPD‐II separately. For BPD‐I patients, the response score in Val/Val group was significantly lower than that in Met allele carriers (t = ?2.27, df = 144, P = 0.025); for BPD‐II patients, the response score in Val/Val group was significantly higher than that in Met allele carriers (t = 2.33, df = 26, P = 0.028). Although these results should be interpreted with caution because of the limited sample for Val/Val genotype in BPD‐II patients (N = 5), these findings strengthen the hypothesis that BDNF pathway gets involved in the etiology and pharmacology of BPD and suggest the differences between BPD‐I and BPD‐II.     

Transport characteristics of a novel peptide platform for CNS therapeutics

New and effective therapeutics that cross the blood‐brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep‐2 peptide, a sequence derived from ligands that bind to low‐density lipoprotein receptor‐related protein‐1 (LRP‐1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep‐2 conjugate and immunohistochemical studies of injected Angiopep‐2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co‐localization with the neuronal nuclei‐selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [¹²⁵I]‐Angiopep‐2 into brain endothelial cells occurred by a saturable mechanism involving LRP‐1. The primary sequence and charge of Angiopep‐2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.