Challenging Case: A Multidisciplinary Approach to Demystifying Chronic Sleep Impairment in an Infant with a Complex Medical and Behavioral Profile.

CASE: X is a 22-month-old White male infant with a complex medical history, including diagnoses of FBXO11 mutation, hypotonia, restrictive lung disease and mild intermittent asthma, laryngotracheomalacia, obstructive sleep apnea (OSA), feeding difficulties with a history of aspiration, gastroesophageal reflux disease (GERD), and developmental delays. X's medical presentation has resulted in multiple prior medical admissions for respiratory failure due to acute illnesses, procedures and treatments including gastrojejunostomy (GJ) tube dependence, supraglottoplasty to reshape tissues of the upper larynx, and the use of biphasic positive airway pressure (BiPAP) at night and room air during the day when he is at baseline. In addition, he has nocturnal events characterized by significant agitation, screaming, crying, body stiffening and limb movements with pauses in breathing, mouth breathing, restless sleep, and difficulty waking in the morning with concomitant daytime fatigue despite above treatments for OSA. There is no history of congenital heart disease or sudden unexplained death. Family history is noncontributory because parents are negative for the FBXO11 variant.X's sleep disruption has led to significant sleep deficits for both X and his caregivers, who spend much of the night strategizing on how to console him. X has undergone several sleep studies, starting when X was aged 4 months, at several children's hospitals across the nation to determine the cause of his chronic sleep disturbance, which yielded limited information and treatment success. As an infant, X received a medical workup and was subsequently treated with a proton pump inhibitor (PPI) for reflux. At 12 months, he was diagnosed with disordered sleep with myoclonic jerks and started on melatonin and gabapentin for involuntary movements. At 13 months, gabapentin was weaned back because of intolerance, and at 15 months, nortriptyline and clonidine were started because of worsening symptoms to target potential neuropathic pain. While most of his symptoms were at night, he had occasional daytime screaming episodes, particularly when experiencing illness. Gabapentin and clonidine were stopped because nortriptyline seemed most effective.At 17 months, the results from a sleep study led to a diagnosis of night terrors, and several clinicians agreed that X's sleep disruption was behavioral in nature. At this time, an infant mental health consultant met with a sleep psychologist on the family's behalf to support family in considering systematic desensitization therapy to increase tolerance to wearing his BiPAP mask, as well as other behavioral and sleep hygiene strategies, which were tried on several occasions and again, resulted in limited improvement in functioning.At 19 months, X's multidisciplinary team reconsidered a night terror diagnosis after a failed trial of clonazepam and pursued a differential diagnosis of periodic limb movement disorder (PLMD). X trialed gabapentin again, but this time only a nighttime dose, per sleep medicine and psychiatry recommendation. While this brought some temporary relief from nighttime distress, despite increasing to the highest dose for age and weight (15 mg/kg/dose), this became less effective, and he was weaned off at 22 months. He had been on iron supplementation since age 6 months and received an iron infusion at 22 months because of persistently low ferritin levels and PLMD in sleep.At 24 months, X was briefly trialed on levetiracetam. While no evidence for seizures on EEG was present, this medication was chosen for involuntary movements and genetic risk for seizures. However, this medication was not useful. At 25 months, an evaluation with a movement disorder physiatrist resulted in a diagnosis of nocturnal paroxysmal dystonia, and he was started on baclofen, which has provided some, but not complete relief to nighttime symptoms. Parents are reporting he has more "good nights" than "bad nights," but "bad nights" come in stretches of a few days in length with no known trigger or relief.Most recently, X was evaluated by general genetics. Whole exome sequencing (WES) was pursued which revealed a pathogenic de novo variant in FBXO11 and provides a likely cause for his neurodevelopmental phenotype. However, he has some features not explained by FBX011; thus, reanalysis of his WES was performed and revealed a de novo variant of uncertain significance in RAF1. Because pathogenic variants in RAF1 have been associated with dilated cardiomyopathy and Noonan spectrum disorder, it was recommended that X be followed periodically in a cardiac genetics clinic. Family is well connected into the FBXO11 community, including supportive Facebook groups. Parents have shared that they do not feel X's breathing issues and pain fit with the phenotype of other children with FBXO11 mutations.X is also enrolled in a medical child care program to facilitate development and social-emotional functioning and receives learning, speech, occupational, physical, and feeding therapy while in attendance. Despite periods of absence due to contracting numerous viral illnesses over the past several months, X continues to make progress across developmental therapies and happily engages when at the program.What additional diagnostic tests and treatment should be considered to better understand X's medical and behavioral presentation? What are the implications of chronic sleep deprivation and stress on the behavior and development of infant with X's profile? What are important psychosocial considerations because it relates to children with medical complexity (CMC), particularly for X and his family to support caregiver, family, and X's quality of life and overall well-being?

FBXO30 functions as a tumor suppressor and an E3 ubiquitin ligase for hZIP1­mediated HIF­1α degradation in renal cell carcinoma.

Studies on clear cell renal cell carcinoma (ccRCC) are gaining momentum due to its high malignancy and potential to metastasize. F­box protein 30 (FBXO30) is a member of the F­box protein family; however, its role and mechanism in cancer remains to be fully elucidated. Western blotting, reverse transcription­quantitative PCR and immunohistochemsitry were performed to detect the expression levels of FBXO30 in ccRCC tissues and adjacent normal tissues. Tumor biological function assays and animal experiments were conducted to clarify the inhibitory effect of FBXO30 on the progression and metastasis of ccRCC. Protein half­life assay, MG132 inhibition assay, immunofluorescence assay and co­immunoprecipitation assay were performed to explore the ubiquitination mechanism of FBXO30 and HIF­1α. Zinc supplementation assay was used to verify the regulatory relationship between human ZRT, IRT­like protein 1 (hZIP1), FBXO30 and HIF­1α. The present study revealed that the expression levels of FBXO30 were lower in ccRCC tissues compared with those in normal adjacent tissues. In addition, FBXO30 inhibited the tumorigenesis and metastatic capacity of ccRCC cells in vivo and in vitro. FBXO30 mediated the ubiquitination and degradation of hypoxia­inducible factor­1α (HIF­1α) in ccRCC cells under normoxia, thereby inhibiting the oncogenic effect of HIF­1α. Notably, hZIP1 served as an upstream regulator of FBXO30, regulating the expression of FBXO30 and HIF­1α by recruiting Zn2+. In conclusion, the present data suggested that FBXO30 is a novel E3 ubiquitination ligase that can function as a tumor suppressor in ccRCC, and the hZIP1/Zn2+/FBXO30/HIF­1α axis may provide potential biomarkers or therapeutic targets for ccRCC.

Promising anticancer activities and mechanisms of action of active compounds from the medicinal herb Centipeda minima (L.) A. Braun & Asch.

BACKGROUND: Centipeda minima (L.) A. Braun & Asch (C. minima) has been used as a traditional Chinese herbal medicine to treat multiple diseases, including sinusitis, rhinitis, headache, and allergy. To date, the anticancer properties of C. minima have drawn considerable attention owing to the anticancer potential of C. minima extracts, the identification of active components, and the elucidation of underlying molecular mechanisms. However, the anticancer properties and significance of active components in C. minima have rarely been summarized. PURPOSE: This review presents a comprehensive summary of the anticancer properties exhibited by active components of C. minima. METHODS: An extensive search for published articles on the anticancer activities and active components of C. minima was performed using Web of Science, PubMed, Science Direct, and Google Scholar. RESULTS: C. minima extracts exhibited both anticancer and chemosensitizing effects. Phytochemical studies have identified the active anticancer components of C. minima extracts. Sesquiterpene lactones, such as 6-O-angeloylplenolin (6-OAP, or brevilin A) and arnicolide D, have similar structures and anticancer mechanisms. As the most abundant sesquiterpene lactone in C. minima, 6-OAP exhibits anticancer activities mainly by targeting Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase and signal transducers and activators of transcription 3 (STAT3). Clinical trials have assessed the potential of 6-OAP in patients with vertex balding and alopecia areata, given its effect on JAK-STATs signaling. Chlorogenic acid, a representative organic acid in C. minima, reportedly possesses anticancer potential and inhibits tumor growth by affecting tumor microenvironment and has been approved for phase II clinical trials in patients with glioma in China. CONCLUSION: In the present review, we highlight intriguing anticancer properties mediated by active compounds isolated from C. minima extracts, particularly sesquiterpene lactones, which might provide clues for developing novel anticancer drugs. Relevant clinical trials on chlorogenic acid and 6-OAP can promote anticancer clinical applications. Therefore, it is worth comprehensively elucidating underlying anticancer mechanisms and conducting clinical trials on C. minima and its active components.

A nonS-locus F-box gene breaks self-incompatibility in diploid potatoes.

Potato is the third most important staple food crop. To address challenges associated with global food security, a hybrid potato breeding system, aimed at converting potato from a tuber-propagated tetraploid crop into a seed-propagated diploid crop through crossing inbred lines, is under development. However, given that most diploid potatoes are self-incompatible, this represents a major obstacle which needs to be addressed in order to develop inbred lines. Here, we report on a self-compatible diploid potato, RH89-039-16 (RH), which can efficiently induce a mating transition from self-incompatibility to self-compatibility, when crossed to self-incompatible lines. We identify the S-locusinhibitor (Sli) gene in RH, capable of interacting with multiple allelic variants of the pistil-specific S-ribonucleases (S-RNases). Further, Sli gene functions like a general S-RNase inhibitor, to impart SC to RH and other self-incompatible potatoes. Discovery of Sli now offers a path forward for the diploid hybrid breeding program.

Antileukemic effect of caffeic acid 3,4-dihydroxyphenetyl ester. Evidences for its mechanisms of action.

BACKGROUND: Caffeic acid 3,4-dihydroxyphenethyl ester (CADPE) is a natural polyphenolic ester isolated as a minor component from a water extract of the Chinese medicine Zhongjiefeng [Sarcandra glabra (Thunb.) Nakai (Chloranthaceae)] and has previously shown to have activity against solid tumors through the modulation of multiple targets or signal pathways. However, the activity and potential mechanism of CADPE against leukemia cells have not yet been characterized. PURPOSE: To investigate whether and how CADPE kills leukemia cells. METHOD: (1) The activity of CADPE inhibiting the growth of different leukemia cell lines was evaluated by MTT assay; (2) Cell cycle arrest and apoptosis induced by CADPE were determined by flow cytometry with FlowJo software for quantification; (3) The protein levels were analyzed by Western blot and ubiquitin-binding c-Myc was acquired by co-immunoprecipitation. RESULTS: CADPE exerted potent activity against different leukemia cell lines with low toxicity in normal cells. In terms of mechanism of action, CADPE promoted ubiquitin-proteasome-dependent degradation of c-Myc through activating glycogen synthase kinase-3ß (GSK3ß) and downregulating deubiquitinating enzyme USP28 to trigger the interaction of c-Myc with ubiquitin ligase Fbw7, resulting in the downregulation of cell cycle regulators and anti-apoptotic proteins and consequently, cell cycle arrest and cell apoptosis. CONCLUSION: CADPE is a novel c-Myc inhibitor with high activity and a unique mechanism for killing leukemia cells.

Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development.

Fruit development is orchestrated by a complex network of interactions between hormone signaling pathways. The phytohormone gibberellin (GA) is known to regulate a diverse range of developmental processes; however, the mechanisms of GA action in perennial fruit species are yet to be elucidated. In the current study, a GA signaling gene PslSLY1, encoding a putative F-box protein that belongs to the SLY1 (SLEEPY1)/GID2 (gibberellin-insensitive dwarf2) gene family, was isolated from Japanese plum (Prunus salicina). PslSLY1 transcript abundance declined as fruit development progressed, along with potential negative feedback regulation of PslSLY1 by GA. Subcellular localization and protein-protein interaction assays suggested that PslSLY1 functions as an active GA signaling component that interacts with the ASK1 (Arabidopsis SKP1) subunit of an SCF-ubiquitin ligase complex and with PslDELLA repressors, in a GA-independent manner. By using a domain omission strategy, we illustrated that the F-box and C-terminal domains of PslSLY1 are essential for its interactions with the downstream GA signaling components. PslSLY1 overexpression in wild-type and Arabidopsissly1.10 mutant backgrounds resulted in a dramatic enhancement in overall plant growth, presumably due to triggered GA signaling. This includes germination characteristics, stem elongation, flower structure, and fertility. Overall, our findings shed new light on the GA strategy and signaling network in commercially important perennial crops.

AaCOI1, Encoding a CORONATINE INSENSITIVE 1-Like Protein of Artemisia annua L., Is Involved in Development, Defense, and Anthocyanin Synthesis.

Artemisia annua is an important medicinal plant producing the majority of the antimalarial compound artemisinin. Jasmonates are potent inducers of artemisinin accumulation in Artemisisa annua plants. As the receptor of jasmonates, the F-box protein COI1 is critical to the JA signaling required for plant development, defense, and metabolic homeostasis. AaCOI1 from Artemisia annua, homologous to Arabidopsis AtCOI1, encodes a F-box protein located in the nuclei. Expressional profiles of the AaCOI1 in the root, stem, leaves, and inflorescence was investigated. The mRNA abundance of AaCOI1 was the highest in inflorescence, followed by in the leaves. Upon mechanical wounding or MeJA treatment, expression of AaCOI1 was upregulated after 6 h. When ectopically expressed, driven by the native promoter from Arabidopsis thaliana, AaCOI1 could partially complement the JA sensitivity and defense responses, but fully complemented the fertility, and the JA-induced anthocyanin accumulation in a coi1-16 loss-of-function mutant. Our study identifies the paralog of AtCOI1 in Artemisia annua, and revealed its implications in development, hormone signaling, defense, and metabolism. The results provide insight into JA perception in Artemisia annua, and pave the way for novel molecular breeding strategies in the canonical herbs to manipulate the anabolism of pharmaceutic compounds on the phytohormonal level.

Tubby-like Protein 2 regulates homogalacturonan biosynthesis in Arabidopsis seed coat mucilage.

KEY MESSAGE: A possible transcription factor TLP2 was identified to be involved in the regulation of HG biosynthesis in Arabidopsis seed mucilage. TLP2 can translocate into nucleus from plasma membrane by interacting with NF-YC3. The discovery of TLP2 gene function can further fulfill the regulatory network of pectin biosynthesis in Arabidopsis thaliana. Arabidopsis seed coat mucilage is an excellent model system to study the biosynthesis, function and regulation of pectin. Rhamnogalacturonan I (RG-I) and homogalacturonan (HG) are the major polysaccharides constituent of the Arabidopsis seed coat mucilage. Here, we identified a Tubby-like gene, Tubby-like protein 2 (TLP2), which was up-regulated in developing siliques when mucilage began to be produced. Ruthenium red (RR) staining of the seeds showed defective mucilage of tlp2-1 mutant after vigorous shaking compared to wild type (WT). Monosaccharide composition analysis revealed that the amount of total sugars and galacturonic acid (GalA) decreased significantly in the adherent mucilage (AM) of tlp2-1 mutant. Immunolabelling and dot immunoblotting analysis showed that unesterified HG decreased in the tlp2-1 mutant. Furthermore, TLP2 can translocate into nucleus by interacting with Nuclear Factor Y subunit C3 (NF-YC3) to function as a transcription factor. RNA-sequence and transactivation assays revealed that TLP2 could activate UDP-glucose 4-epimerase 1 (UGE1). In all, it is concluded that TLP2 could regulate the biosynthesis of HG possibly through the positive activation of UGE1.

S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen.

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific S-RNase gene and multiple pollen-specific S-locus F-box (SLF) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/Cas9 to generate two frame-shift indel alleles of S2 -SLF1 (SLF1 of S2 -haplotype) in S2S3 plants of P. inflata and examined the effect on the SI behavior of S2 pollen. In the absence of a functional S2-SLF1, S2 pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S2 -haplotype and these eight S-RNases that had been determined by gain-of-function experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility.

Ex vivo lung perfusion as a human platform for preclinical small molecule testing.

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation.

Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron-sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.

FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation.

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7-inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA or p53. These cells are defective in apoptosis owing to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug-resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms.

Medicinal herbs Oenanthe javanica (Blume) DC., Casuarina equisetifolia L. and Sorghum bicolor (L.) Moench protect human cells from MPP+ damage via inducing FBXO7 expression.

BACKGROUND: The F-box protein 7 (FBXO7) mutations have been identified in families with early-onset parkinsonism and pyramidal tract signs, and designated as PARK15. In addition, FBXO7 mutations were found in typical and young onset Parkinson's disease (PD). Evidence has also shown that FBXO7 plays an important role in the development of dopaminergic neurons and increased stability and overexpression of FBXO7 may be beneficial to PD. PURPOSE: We screened extracts of medicinal herbs to enhance FBXO7 expression for neuroprotection in MPP+-treated cells. METHODS: Promoter reporter assay in HEK-293 cells was used to examine the cis/trans elements controlling FBXO7 expression and to screen extracts of medicinal herbs enhancing FBXO7 expression. MTT assay was performed to assess cell viability of MPP+-treated HEK-293/SH-SY5Y cells. In addition, proteasome activity, mitochondrial membrane potential and FBXO7/TRAF2/GATA2 protein expression were evaluated. RESULTS: We demonstrated that -202--57 region of the FBXO7 promoter is likely to contain sequences that are bound by positive trans protein factors to activate FBXO7 expression and GATA2 is the main trans protein factor enhancing FBXO7 expression. Extracts of medicinal herbs Oenanthe javanica (Blume) DC. (Umbelliferae), Casuarina equisetifolia L. (Casuarinaceae), and Sorghum bicolor (L.) Moench (Gramineae) improved cell viability of both MPP+-treated HEK-293 and SH-SY5Y cells, rescued proteasome activity in MPP+-treated HEK-293 cells, and restored mitochondrial membrane potential in MPP+-treated SH-SY5Y cells. These protection effects of herbal extracts are acting through enhancing FBXO7 and decreasing TRAF2 expression, which is probably mediated by GATA2 induction. CONCLUSION: Collectively, our study provides new targets, FBXO7 and its regulator GATA2, for the development of potential treatments of PD.

All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility.

Recognition of a wide-range of S-RNases by S locus F-box like 2, a general-inhibitor candidate in the Prunus-specific S-RNase-based self-incompatibility system.

Many species in the Rosaceae, the Solanaceae, and the Plantaginaceae exhibit S-RNase-based gametophytic self-incompatibility (GSI). This system comprises S-ribonucleases (S-RNases) as the pistil S determinant and a single or multiple F-box proteins as the pollen S determinants. In Prunus, pollen specificity is determined by a single S haplotype-specific F-box protein (SFB). The results of several studies suggested that SFB exerts cognate S-RNase cytotoxicity, and a hypothetical general inhibitor (GI) is assumed to detoxify S-RNases in non-specific manner unless it is affected by SFB. Although the identity of the GI is unknown, phylogenetic and evolutionary analyses have indicated that S locus F-box like 1-3 (or S locus F-box with low allelic sequence polymorphism 1-3; SLFL1-3), which are encoded by a region of the Prunus genome linked to the S locus, are good GI candidates. Here, we examined the biochemical characteristics of SLFL1-3 to determine whether they have appropriate GI characteristics. Pull-down assays and quantitative expression analyses indicated that Prunus avium SLFL1-3 mainly formed a canonical SCF complex with PavSSK1 and PavCul1A. Binding assays with PavS(1,3,4,6)-RNases showed that PavSLFL1, PavSLFL2, and PavSLFL3 bound to PavS(3)-RNase, all PavS-RNases tested, and none of the PavS-RNases tested, respectively. Together, these results suggested that SLFL2 has the appropriate characteristics to be the GI in sweet cherry pollen, while SLFL1 may redundantly work with SLFL2 to detoxify all S-RNases. We discuss the possible roles of SLFL1-3 as the GI in the Prunus-specific S-RNase-based GSI mechanism.

Increased level of reactive oxygen species persuades postovulatory aging-mediated spontaneous egg activation in rat eggs cultured in vitro.

The present study was aimed to find out whether increased level of reactive oxygen species (ROS) particularity hydrogen peroxide (H2O2) could persuade postovulatory aging-mediated abortive spontaneous egg activation (SEA) in rat eggs cultured in vitro. For this purpose, ROS and H2O2 levels, mitochondria distribution and its membrane potential, p286-CaMK-II, Emi2, Thr-161 phophorylated cyclin-dependent protein kinase1 (Cdk1) as well as cyclin B1 levels, in vitro effects of 3-tert-butyl-4 hydroxy anisole (BHA), pentoxifylline and dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) were analyzed during postovulatory aging-induced abortive SEA in vitro. Data of the present study suggest that postovulatory aging increased H2O2 levels, disturbed mitochondrial distribution pattern and mitochondrial membrane potential (MMP) in eggs. There was an significant increase of p286-CaMK-II level, while Emi2 level reduced significantly during egg aging in vitro. The reduced Emi2 level was associated with decreased Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 level in aged eggs that underwent abortive SEA. Further, supplementation of pentoxifylline, db-cAMP, and BHA protected postovulatory aging-mediated abortive SEA in concentration-dependent manner. These data suggest that postovulatory aging increased H2O2 levels, reduced MMP, and increased p286-CaMK-II. The increased p286-CaMK-II was associated with reduced Emi2 level and maturation-promoting factor levels during postovulatory aging-mediated abortive SEA. Drugs that elevate cAMP directly or indirectly and BHA protected postovulatory aging-mediated abortive SEA possibly by reducing ROS level in rat eggs cultured in vitro.

Genome-wide analysis of phylogeny, expression profile and sub-cellular localization of SKP1-Like genes in wild tomato.

SKP1 is a core component of SCF complex, a major type of E3 ubiquitin ligase catalyzing the last step in ubiquitin-mediated protein degradation pathway. In present study, SKP1 gene family in Solanum pimpinellifolium (SSK), a wild species of tomato, was investigated. A total of 19 SSK genes were identified through homologous search. Their chromosomal locations, gene structures, phylogeny, expression profiles, sub-cellular localizations and protein-protein interaction patterns with putative F-box proteins were analyzed in detail. The high homology and similar expression patterns among clustered SSK genes in chromosome suggested that they may have evolved from duplication events and are functionally redundant. Sub-cellular localization indicated that most of the SSK proteins are distributed in both cytosol and nucleus, except for SSK8, which is detected in cytosol only. Tissue-specific expression patterns suggested that many SSK genes may be involved in tomato fruit development. Furthermore, several SSK genes were found to be responsive to heat stress and salicylic acid treatment. Based on phylogenetic analysis, expression profiles and protein interaction property, we proposed that tomato SSK1 and SSK2 might have similar function to ASK1 and ASK2 in Arabidopsis.