RGF1 controls root meristem size through ROS signalling.

The stem cell niche and the size of the root meristem in plants are maintained by intercellular interactions and signalling networks involving a peptide hormone, root meristem growth factor 1 (RGF1)1. Understanding how RGF1 regulates the development of the root meristem is essential for understanding stem cell function. Although five receptors for RGF1 have been identified2-4, the downstream signalling mechanism remains unknown. Here we report a series of signalling events that follow RGF1 activity. We find that the RGF1-receptor pathway controls the distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a previously uncharacterized transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), that has a central role in mediating RGF1 signalling. Manipulating RITF1 expression leads to the redistribution of ROS along the root developmental zones. Changes in ROS distribution in turn enhance the stability of the PLETHORA2 protein, a master regulator of root stem cells. Our results thus clearly depict a signalling cascade that is initiated by RGF1, linking this peptide to mechanisms that regulate ROS.

PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Root Canal Treatment of Oehlers Type III Dens Invaginatus in Maxillary Lateral Incisor and Remote Sinus Tract Using Dental Surgical Microscope and Cone-Beam Computed Tomography.

Dens invaginatus is a morphological abnormality of the tooth that results from a developmental anomaly during tooth formation, in which part of the enamel and dentin of the crown invaginates into the pulp cavity. This report describes a case of a maxillary lateral incisor with apical periodontitis apparently caused by Oehlers Type III dens invaginatus. The patient was a 69-year-old man who visited our clinic complaining of discomfort in the maxillary right lateral incisor. Cone-beam computed tomography (CBCT) revealed dens invaginatus of the maxillary lateral incisor and a sinus tract in the maxillary central incisor region, which was derived from apical periodontitis of the maxillary lateral incisor. The dens invaginatus was accompanied by a complex root canal morphology. Treatment, which was performed using a dental surgical microscope, had a favorable outcome. The patient remains in good condition at 1 year postoperatively.

Comparison of Characteristics of Dental Malpractice Trials between Medical Malpractice and Ordinary Divisions in District Courts.

The purpose of this study was to elucidate the characteristics of dental malpractice trials undertaken by medical malpractice divisions and ordinary divisions in district courts. Dentistry disputes in a total of 84 trials held between 1977 and 2014 were investigated. A total of 45 were conducted by medical malpractice divisions, resulting in 18 approvals and 27 dismissals, while 39 were undertaken by ordinary divisions, leading to 24 approvals and 15 dismissals. The parameters analyzed comprised category of dental treatment, judgment, amount claimed, and amount accepted. The results revealed that the mean amount claimed in trials held by medical malpractice divisions (¥12,563,324) was lower than that sought in trials conducted by ordinary divisions. The amount accepted was also found to exceed 50% of the amount claimed in 6 trials held by ordinary divisions (maximum 75.2%), but in only 2 trials conducted by medical malpractice divisions (maximum 54.8%); the mean amount accepted in trials held by medical malpractice divisions was 24.2%. These results indicate that judgments in trials conducted by medical malpractice divisions place a stronger emphasis on resolution of dental malpractice dispute than on pursuing truth or carrying out proper legal procedures.

Intentional Replantation to Treat Apical Periodontitis of Maxillary First Molar with Foreign Body Located Outside Apical Foramen Using CBCT: A Case Report.

The recent use of cone beam computed tomography (CBCT) in the field of dentistry to obtain 3-dimensional (3D) images has enabled more effective examination and diagnosis in endodontic treatment. Such information has also been reported to be of benefit in surgical endodontic treatment such as intentional replantation. Here, we report a case of intentional replantation with the assistance of CBCT in which a good therapeutic outcome was achieved. The patient was a 30-year-old woman who visited our hospital with the chief complaint of spontaneous pain in the right maxillary first molar. Dental radiographs revealed a radiolucent area in the apical portion of the distal root, and a radiopaque area thought to be a broken shard from a small surgical instrument measuring approximately 1.5 mm in length. The shard was located outside the distal apical foramen. Based on these findings, the diagnosis was acute suppurative apical periodontitis of the right maxillary first molar. Infected root canal therapy was subsequently commenced. The patient's symptoms showed no improvement, however, and the pain persisted. Therefore, dental CBCT was performed to obtain 3D images, which confirmed a radiopaque area thought to be a broken shard from a small surgical instrument located outside the apical foramen of the distal root and facing in a direction that made it impossible to remove from the root canal. Because the patient's symptoms had shown no improvement and a foreign body was observed outside the apical foramen, intentional replantation combined with root resection was performed with informed consent. At a 1-year follow-up visit, progress was good, and there were no reported symptoms or signs. When reaching a diagnosis is difficult based on clinical findings and dental radiographs alone, the 3D images provided by CBCT offer a means of securing a more reliable diagnosis, allowing planning of treatment to be more effective.

A plant U-box protein, PUB4, regulates asymmetric cell division and cell proliferation in the root meristem.

The root meristem (RM) is a fundamental structure that is responsible for postembryonic root growth. The RM contains the quiescent center (QC), stem cells and frequently dividing meristematic cells, in which the timing and the frequency of cell division are tightly regulated. In Arabidopsis thaliana, several gain-of-function analyses have demonstrated that peptide ligands of the Clavata3 (CLV3)/embryo surrounding region-related (CLE) family are important for maintaining RM size. Here, we demonstrate that a plant U-box E3 ubiquitin ligase, PUB4, is a novel downstream component of CLV3/CLE signaling in the RM. Mutations in PUB4 reduced the inhibitory effect of exogenous CLV3/CLE peptide on root cell proliferation and columella stem cell maintenance. Moreover, pub4 mutants grown without exogenous CLV3/CLE peptide exhibited characteristic phenotypes in the RM, such as enhanced root growth, increased number of cortex/endodermis stem cells and decreased number of columella layers. Our phenotypic and gene expression analyses indicated that PUB4 promotes expression of a cell cycle regulatory gene, CYCD6;1, and regulates formative periclinal asymmetric cell divisions in endodermis and cortex/endodermis initial daughters. These data suggest that PUB4 functions as a global regulator of cell proliferation and the timing of asymmetric cell division that are important for final root architecture.

Functions of long intergenic non-coding (linc) RNAs in plants.

Whole transcriptome analyses in many organisms have revealed that most transcribed RNAs do not encode proteins. These non-coding RNAs likely contribute to the regulation of gene expression during the development of multicellular organisms. In eukaryotes, the roles of small RNAs, one class of non-coding RNAs, in transcriptional and post-transcriptional regulation have been well characterized. However, the functions of a second class of non-coding RNAs, long intergenic noncoding (linc) RNAs, are relatively unknown, especially in plants. Recent advances in RNA-seq and tiling microarray technologies have revealed the presence of many lincRNAs across plant species. This review focuses on the functions of lincRNAs that have been recently reported in plants. One of the most well characterized functions of lincRNAs is to epigenetically regulate gene expression by recruiting proteins for chromosome modification to specific loci. Second, lincRNAs are known to inhibit the physical interaction between microRNAs (miRNAs) and their target mRNAs thus controling protein levels of the target mRNAs. Lastly, lincRNAs control alternative splicing by binding and sequestering the proteins required for alternative splicing.

Heterotrimeric G proteins control stem cell proliferation through CLAVATA signaling in Arabidopsis.

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gß mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.

BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems.

Molecular Basis of Laminin-Integrin Interactions.

Laminins are composed of three polypeptide chains, designated as α, ß, and γ. The C-terminal region of laminin heterotrimers, containing coiled-coil regions, short tails, and laminin globular (LG) domains, is necessary and sufficient for binding to integrins, which are the major laminin receptor class. Laminin recognition by integrins critically requires the α chain LG domains and a glutamic acid residue of the γ chain at the third position from the C-terminus. Furthermore, the C-terminal region of the ß chain contains a short amino acid sequence that modulates laminin affinity for integrins. Thus, all three of the laminin chains act cooperatively to facilitate integrin binding. Mammals possess 5 α (α1-5), 3 ß (ß1-3), and 3 γ (γ1-3) chains, combinations of which give rise to 16 distinct laminin isoforms. Each isoform is expressed in a tissue-specific and developmental stage-specific manner, exerting its functions through binding of integrins. In this review, we detail the current knowledge surrounding the molecular basis and physiological relevance of specific interactions between laminins and integrins, and describe the mechanisms underlying laminin action through integrins.

Processive phosphorylation of ERK MAP kinase in mammalian cells.

The mitogen-activated protein (MAP) kinase pathway is comprised of a three-tiered kinase cascade. The distributive kinetic mechanism of two-site MAP kinase phosphorylation inherently generates a nonlinear switch-like response. However, a linear graded response of MAP kinase has also been observed in mammalian cells, and its molecular mechanism remains unclear. To dissect these input-output behaviors, we quantitatively measured the kinetic parameters involved in the MEK (MAPK/ERK kinase)-ERK MAP kinase signaling module in HeLa cells. Using a numerical analysis based on experimentally determined parameters, we predicted in silico and validated in vivo that ERK is processively phosphorylated in HeLa cells. Finally, we identified molecular crowding as a critical factor that converts distributive phosphorylation into processive phosphorylation. We proposed the term quasi-processive phosphorylation to describe this mode of ERK phosphorylation that is operated under the physiological condition of molecular crowding. The generality of this phenomenon may provide a new paradigm for a diverse set of biochemical reactions including multiple posttranslational modifications.

Critical Factors Affecting Outcomes of Endodontic Microsurgery: A Retrospective Japanese Study.

The critically important preoperative and intraoperative factors that affect the success of endodontic microsurgery (EMS) in Japanese patients are not defined. We conducted a retrospective study that analyzed treatment outcomes for 46 teeth in 46 Japanese patients. Treatment was provided between March 2013 and March 2015. All patients were evaluated after one year, the shortest time period over which treatment outcomes after apicoectomy could be evaluated and in which there were complete records for the recruited patient population. Healing was assessed on the basis of clinical symptoms and radiographs. With the use of a binary logistic regression model to quantify success, we estimated the effects of patient age, sex, dental arch, lesion size, lesion type, preoperative root canal treatment, the presence or absence of a post core, and the presence or absence of an isthmus on the surgically prepared dentine surface. The overall success for EMS was 93.5% after one year; failures comprised 6.5%. Successful outcomes were higher (p = 0.04) for maxillary teeth than for mandibular teeth. Success was higher (p = 0.019) for patients who received root canal instrumentation prior to EMS. Age, sex, lesion size, lesion type, the presence or absence of a post core, and the presence or absence of a root canal isthmus had no effect (p > 0.2) on success. We conclude that the percentage of successful outcomes after EMS treatment for Japanese patients presenting with periapical periodontitis is very high after one year and that success is influenced strongly by the dental arch and preoperative root canal instrumentation.

Disease-associated single amino acid mutation in the calf-1 domain of integrin α3 leads to defects in its processing and cell surface expression.

Integrin α3ß1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human α3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of α3. Specifically, α3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized α3. The mutant α3 retained the ability to associate with integrin ß1, but not with the tetraspanin CD151, and the bound ß1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of α3ß1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of α3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of α3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the ß-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of α3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.

New Insights on the Role of ß-Cyanoalanine Synthase CAS-C1 in Root Hair Elongation through Single-Cell Proteomics.

(1) Background: Root hairs are specialized structures involved in water and plant nutrient uptake. They elongate from epidermal cells following a complex developmental program. ß-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. (2) Methods: To identify its role in root hair formation, we conducted single-cell proteomics analysis by isolating root hair cells using Fluorescence-activated Cell Sorting (FACS) from wild-type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity in proteins of wild type and cas-c1 mutants. (3) Results and Conclusions: We have found that the cas-c1 mutation has no visible effect on quiescent center or meristem root tissue, in both control and nutrient-deprivation conditions. We have identified more than 3900 proteins in root hairs and we have found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and DNA methylation, are modified by S-cyanylation.

NAC103 mutation alleviates DNA damage in an Arabidopsis thaliana mutant sensitive to excess boron.

Excess boron (B) is toxic to plants and thereby causes DNA damage and cell death in root meristems. However, the underlying mechanisms which link boron and DNA damage remain unclear. It has been reported that the rpt5a-6 mutant of the 26S proteasome is sensitive to excess boron, resulting in more frequent cell death in root meristem and reduced root elongation. In this study, we showed that a reduction in root growth in the rpt5a mutant in the presence of high boron levels is repressed by a mutation in NAC domain containing transcription factor NAC103, a substrate of the proteasome, which functions in the unfolded protein response pathway. The mutation in NAC103 alleviated excess-B-induced DNA damage and cell death in root meristems of the rpt5a mutant. Superoxide ( O 2 - ) staining with nitroblue tetrazolium revealed that boron stress causes O 2 - accumulation in root tips, which was higher in the rpt5a-6 mutant, whereas the accumulation was lower in the rpt5a-6 nac103-3 double mutant. Our work demonstrates the overall involvement of NAC103 in maintaining healthy root meristem under excess boron conditions in the absence of RPT5A proteasome subunit.

Luminescence imaging of water irradiated by protons under FLASH radiation therapy conditions.

Objective.FLASH radiation therapy with ultrahigh dose rates (UHDR) has the potential to reduce damage to normal tissue while maintaining anti-tumor efficacy. However, rapid and precise dose distribution measurements remain difficult for FLASH radiation therapy with proton beams. To solve this problem, we performed luminescence imaging of water following irradiation by a UHDR proton beam captured using a charge-coupled device camera.Approach. We used 60 MeV proton beams with dose rates of 0.03-837 Gy s-1from a cyclotron. Therapeutic 139.3 MeV proton beams with dose rates of 0.45-4320 Gy s-1delivered by a synchrotron-based proton therapy system were also tested. The luminescent light intensity induced by the UHDR beams was compared with that produced by conventional beams to compare the dose rate dependency of the light intensity and its profile.Main results. Luminescence images of water were clearly visualized under UHDR conditions, with significantly shorter exposure times than those with conventional beams. The light intensity was linearly proportional to the delivered dose, which is similar to that of conventional beams. No significant dose-rate dependency was observed for 0.03-837 Gy s-1. The light-intensity profiles of the UHDR beams agreed with those of conventional beams. The results did not differ between accelerators (synchrotron or cyclotron) and beam energies.Significance. Luminescence imaging of water is achievable with UHDR proton beams as well as with conventional beams. The proposed method should be suitable for rapid and easy quality assurance investigations for proton FLASH therapy, because it facilitates real-time, filmless measurements of dose distributions, and is useful for rapid feedback.

Effectiveness and Cost-effectiveness of Online Brief Mindfulness-based Cognitive Therapy for the Improvement of Productivity in the Workplace: Study Protocol for a Randomized Controlled Trial.

BACKGROUND: Numerous studies have demonstrated the effectiveness of mindfulness-based programs (MBPs) among both clinical and nonclinical populations. These data document positive impacts in the workplace, including reducing perceived stress and burnout and increasing well-being. However, the effectiveness for productivity, which is of most interest to managers and administrators, is still unclear. In addition, MBPs in the workplace tend to be modified by reducing the number of the program sessions or delivering content online to improve accessibility. To date, however, the impact of MBPs that feature these modifications on productivity in the workplace has not been investigated. OBJECTIVE: The study aims to investigate the effectiveness and cost-effectiveness of online-delivered brief mindfulness-based cognitive therapy (bMBCT) for improving productivity and other work-related outcomes among healthy workers compared to the waitlist control. METHODS: We will conduct a 4-week randomized controlled trial (RCT) with a 6-month follow-up. Employees are included in the study if they (1) are between the ages of 20 and 65 years and (2) work longer than 30 hours weekly. Employees are randomly allocated to either the bMBCT group or the waitlist control group. The primary outcome of the study is the mean difference of productivity measured by the World Health Organization Health and Work Performance Questionnaire (WHO-HPQ) between the groups at 4, 16, and 28 weeks. Secondary outcomes include several clinical outcomes and health economics evaluation. RESULTS: We started recruiting participants in August 2021, and the intervention began in October 2021. A total of 104 participants have been enrolled in the study as of October 2021. The intervention is scheduled to be completed in December 2023. Data collection will be completed by the end of January 2024. CONCLUSIONS: The novelty of the study is that (1) it will investigate bMBCT's effectiveness on productivity, which is still unclear, and (2) samples are recruited from 3 companies in different industries. The limitations of the study are that (1) all measures assessed are in self-report format and (2) we lack an active control group. This study has the potential to provide new data on the relationship between MBPs and occupational health and productivity. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry UMIN000044721; https://tinyurl.com/4e2fh873. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/36012.

Effectiveness of Mindfulness-Based Cognitive Therapy With Follow-Up Sessions for Pharmacotherapy-Refractory Anxiety Disorders: Protocol for a Feasibility Randomized Controlled Trial.

BACKGROUND: Augmented mindfulness-based cognitive therapy (MBCT) with treatment as usual (mainly pharmacotherapy) is reported to be effective after treatment for anxiety disorders. However, whether its effectiveness persists in the long term is unclear. OBJECTIVE: This study aims to examine the feasibility, acceptability, and effectiveness of a follow-up program by conducting a feasibility randomized controlled trial (RCT) that compares augmented MBCT with follow-up sessions and that without follow-up sessions in preparation for a definitive RCT. METHODS: The study involves an 8-week MBCT with a 10-month follow-up. Patients aged 20 to 65 years who meet the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria for panic disorder, agoraphobia, or social anxiety disorder, which is not remitted with usual treatment for at least 4 weeks, will be included in the study and randomly allocated to receive augmented MBCT with follow-up sessions or augmented MBCT without follow-up sessions. For this feasibility RCT, the primary outcomes are (1) study inclusion rate, (2) dropout rate, (3) attendance rate, and (4) mean and standard deviation of several clinical measures at 8 weeks and 5, 8, and 12 months. RESULTS: We started recruiting participants in January 2020, and 43 participants have been enrolled up to January 2021. The study is ongoing, and data collection will be completed by May 2022. CONCLUSIONS: This study is novel in terms of its design, which compares augmented MBCT with and without follow-up sessions. The limitations of the trial are as follows: (1) mixed participants in terms of the delivery mode of the intervention, and (2) lack of a pharmacotherapy-alone arm. Owing to its novelty and significance, this study will provide fruitful knowledge for future definitive RCTs. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000038626; https://tinyurl.com/2p9dtxzh. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/33776.

BIT/SHPS-1 promotes antiapoptotic effect of BDNF on low potassium-induced cell death of cultured cerebellar granule neurons.

Brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is a neuronal adhesion molecule that is highly expressed in cerebellar granule neurons (CGNs); however its function in CGNs remains unclear. Our previous studies indicated that BIT/SHPS-1 is able to modulate the antiapoptotic effect of brain-derived neurotrophic factor (BDNF) on CNS neurons by cell type-specific mechanisms. In this article, we have studied the role of BIT/SHPS-1 in the antiapoptotic function of BDNF on low potassium (LK)-induced cell death of cultured CGNs which is an in vitro model system of neuronal apoptosis during brain development. Cultured rat CGNs were transduced with wild-type rat BIT/SHPS-1 (BIT/SHPS-1(WT)), its 4F-mutant (BIT/SHPS-1(4F), in which all cytoplasmic tyrosine residues were substituted with phenylalanine), or nuclear localization signal-attached beta-galactosidase (NLS-LacZ, as control)-expressing adenoviruses. Expression of BIT/SHPS-1(WT) and BIT/SHPS-1(4F) alone did not affect steady-state cell viability. Tyrosine phosphorylation of BIT/SHPS-1 was only detected in BIT/SHPS-1(WT)-expressing cultures in the presence and the absence of BDNF. When subjected to LK in the presence of BDNF, BIT/SHPS-1(WT)- and BIT/SHPS-1(4F)-expressing cultures showed a significant resistance to cell death, while the control virus-transfected culture did not. In addition, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, attenuated the antiapoptotic effect of BDNF on BIT/SHPS-1(WT)-, and BIT/SHPS-1(4F)-expressing cultures. These results demonstrated that in both tyrosine phosphorylation-independent and PI3-K-dependent manners, BIT/SHPS-1 promotes the antiapoptotic effect of BDNF on the LK-induced cell death of CGNs.