Multiresponsive luminescent metal-organic framework for cooking oil adulteration detection and gallium(III) sensing.

A new 3D metal-organic framework {[Cd16(tr2btd)10(dcdps)16(H2O)3(EtOH)]∙15DMF}n (MOF 1, tr2btd = 4,7-di(1,2,4-triazol-1-yl)benzo-2,1,3-thiadiazole, H2dcdps = 4,4'-sulfonyldibenzoic acid) was obtained and its luminescent properties were studied. MOF 1 exhibited bright blue-green luminescence with a high quantum yield of 74 % and luminescence quenching response to a toxic natural polyphenol gossypol and luminescence enhancement response to some trivalent metal cations (Fe3+, Cr3+, Al3+ and Ga3+). The limit of gossypol detection was 0.20 µM and the determination was not interfered by the components of the cottonseed oil. The limit of detection of gallium(III) was 1.1 µM. It was demonstrated that MOF 1 may be used for distinguishing between the genuine sunflower oil and oil adulterated by crude cottonseed oil through qualitative luminescent and quantitative visual gossypol determination.

5-Hydroxymethylfurfural Ameliorates Allergic Inflammation in HMC-1 Cells by Inactivating NF-κB and MAPK Signaling Pathways.

Allergic inflammation is the foundation of multiple allergic disorders, such as allergic rhinitis and asthma. Mast cells are effector cells that initiate inflammatory response. 5-hydroxymethylfurfural (5-HMF), a furfural compound, is the heat-processed product of various fruit, foods, drinks, as well as some Chinese herbal medicines. 5-HMF was previously reported to inhibit mast cell activation. Our study aimed to explore the functions of 5-HMF in both phorbol 12-mystate 13-acetate (PMA) plus calcium ionophore (A23187)-induced allergic inflammation in human mast cell line HMC-1 and ovalbumin (OVA)-induced asthma mouse models. HMC-1 cells were pretreated with 5-HMF and then stimulated by PMA+A23187. The cytotoxicity of 5-HMF on HMC-1 cells was evaluated by MTT assay. Histamine content in cell supernatants was measured by the o-phthaldialdehyde spectrofluorometric procedure. Intracellular calcium was determined using the fluorescent dye Fura-2AM. The production and expression of pro-inflammatory cytokines were detected by ELISA and RT-qPCR. Caspase-1 colorimetric assay was employed to examine the enzymatic activity of caspase-1. Asthma mouse models were induced by OVA sensitization. The bronchoalveolar lavage fluid (BALF) and blood samples were collected for the detection of total and differential cell count as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), OVA-immunoglobulin E (OVA-IgE), OVA-immunoglobulin G1 (OVA-IgG1), and pro-inflammatory cytokine levels. The left lung of mouse was dissected for histopathological examination by hematoxylin and eosin (H&E) staining. The protein expression of pro-caspase-1 and the phosphorylation of NF-κB and MAPK pathway-associated molecules were assessed by Western blotting. Our findings revealed that 5-HMF efficiently suppressed the PMA+A23187-induced enhancement in histamine production and intracellular calcium in HMC-1 cells. Pro-inflammatory cytokine production and expression in HMC-1 cells were elevated after PMA plus A23187 stimulation, which, however, were inhibited by pretreatment of 5-HMF. Additionally, 5-HMF suppressed the activity of caspase-1 and the phosphorylation of NF-κB and MAPK-associated molecules including p65 NF-κB, p38 MAPK, ERK, and JNK in HMC-1 cells. In vivo experiments demonstrated that 5-HMF treatment reduced the lung/body weight index and total and differential (macrophages, neutrophils, lymphocytes, and eosinophils) cell counts in BALF of asthmatic mice, but exerted no influence on serum AST and ALT levels. Besides, 5-HMF reduced serum OVA-IgE and OVA-IgG1 levels, mitigated lung inflammation, and inhibited the NF-κB and MAPK signaling pathways in asthma mouse models. 5-HMF mitigates allergic inflammation in asthma by inactivating caspase-1 and NF-κB and MAPK signaling pathways.

Hypothalamic subregion abnormalities are related to body mass index in patients with sporadic amyotrophic lateral sclerosis.

OBJECTIVE: To investigate atrophy patterns in hypothalamic subunits at different stages of ALS and examine correlations between hypothalamic subunit volume and clinical information. METHODS: We used the King's clinical staging system to divide 91 consecutive ALS patients into the different disease stages. We investigated patterns of hypothalamic atrophy using a recently published automated segmentation method in ALS patients and in 97 healthy controls. We recorded all subjects' demographic and clinical information. RESULTS: Compared with healthy controls, we found significant atrophy in the bilateral anterior-superior subunit and the superior tubular subunit, as well as a reduction in global hypothalamic volume in ALS patients. When we used the King's clinical staging system to divide patients into the different disease stages, we found neither global nor specific subunit atrophy until King's stage 3 in the hypothalamus. Moreover, specific subunit volumes were significantly associated with body mass index. CONCLUSIONS: In a relatively large sample of Chinese patients with ALS, using a recently published automated segmentation method for the hypothalamus, we found the pattern of hypothalamic atrophy in ALS patients differed greatly across King's clinical disease stages. Moreover, specific hypothalamic subunit atrophy may play an important role in energy metabolism in ALS patients. Thus, our findings suggest that hypothalamic atrophy may have potential phenotypic associations, and improved energy metabolism may become an important component of individualised therapy for ALS.

Yangyin Yiqi Mixture Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats through Inhibiting TGF-ß1/Smad Pathway and Epithelial to Mesenchymal Transition.

OBJECTIVE: The aim of the current study was to investigate the protective effect of Yangyin Yiqi Mixture (YYYQ) on Bleomycin-induced pulmonary fibrosis in rats based on TGF-ß1/Smad signal pathway and epithelial to mesenchymal transition (EMT). METHODS: 120 Wistar rats were randomly divided into six groups: control group, BLM group, BLM + Pred group, BLM+YYYQ-L group, BLM+YYYQ-M group, and BLM+YYYQ-H group. Rats were given an intratracheal instillation of 3 mg/kg BLM to establish the pulmonary fibrosis model and followed by different dosages of YYYQ (11, 22, 44g/kg, via intragastric gavage) or prednisone soluble (4.2mg/kg, via intragastric gavage) or water. After 14 days and 28 days, tissue sections were stained with hematoxylin-eosin and Masson's trichrome to observe histopathological changes. Protein levels of TGF-ß1, CTGF, Interleukin 18, and hydroxyproline were detected by ELISA method, and mRNA expressions of TGF-ß1, TßRI, TßRII, Smad3, Smad7, α-SMA, E-cadherin, laminin, and collagen I were detected by RT-PCR. RESULTS: TGF-ß1, CTGF, Interleukin 18, and hydroxyproline levels and mRNA expression of TGF-ß1, TßRI, TßRII, Smad3, α-SMA, laminin, and collagen I were significantly increased (p <0.01), while Smad7 and E-cadherin levels were significantly decreased in BLM group (p <0.01). YYYQ-M and YYYQ-H group had downregulated the TGF-ß1, CTGF, hydroxyproline contents, and mRNA expression of TGF-ß1, TßRI, TßRII, Smad3, α-SMA, laminin, and collagen I and upregulated mRNA levels of Smad7 and E-cadherin significantly (p <0.01 or p <0.05). The result from the present study, which was also supported by histological evidence, suggested that YYYQ-M group and YYYQ-H group exhibited better treatment effect on Bleomycin-induced pulmonary fibrotic rats when compared to that of BLM + Pred group (p <0.01). Meanwhile, the effect of YYYQ, in three different dosages, on the level of interleukin 18 was not significant. CONCLUSION: These results showed that YYYQ has the potential of ameliorating the progression of pulmonary fibrosis, and the mechanism may be related to suppressing TGF-ß1/Smad signal pathway and EMT in BLM-induced pulmonary fibrosis of rats.

Epidemiology study of pediatric primary intussusception aged ≤24 months in pre-rotavirus vaccine era of Jinan, China.

INTRODUCTION: Few studies have reported on the epidemiological characteristics of pediatric primary intussusception in the pre-rotavirus vaccine era of China. It is important to complementary baseline data before rotavirus vaccine introduction in China. This study conduct a retrospective investigation and evaluated the incidence rate, described the epidemiology of pediatric primary intussusception aged ≤24 months. METHODS: We conducted a retrospective investigation in all secondary- and tertiary-hospitals in Jinan. Pediatric primary intussusception inpatients aged ≤24 months were identified depending on ICD-10 discharge code from a total of 63 hospitals from 2011 to 2015. Demographic and clinical information were extracting from the electronic clinical record systems. RESULTS: A total of 575 pediatric primary intussusception inpatients were identified with average annual incidence of 86.5 per 100,000. A significantly higher incidence was observed in males (χ2 = 13.8, P < 0.01), in the ≤12 months old age group (χ2 = 19.5, P < 0.01) and from the urban areas (χ2 = 63.31, P < 0.001). No clear seasonality found. Abdominal pain (80.9%) and vomiting (63.3%) were the most frequently reported. Most intussusception cases occurred in ileo-cecum. Over 92% of intussusception cases were diagnosed by ultrasound alone and 77.4% was successfully treated by air enema. 99.7% were cured. The median time of hospitalization was 2 days (range: 0-35 days). CONCLUSION: This retrospective study provides baseline information of incidence, epidemiologyand clinical characteristics of pediatric primary intussusception in Jinan City during 2011-2016 before the introduction of rotavirus vaccine. It will be important for evaluating safety of rotavirus vaccine if it will be introduced to the routine immunization program in China.

The Influence of BuqiHuoxueTongluo Formula on Histopathology and Pulmonary Function Test in Bleomycin-Induced Idiopathic Pulmonary Fibrosis in Rats.

BuqiHuoxueTongluo Formula (BHTF) is an effective herbal prescription based on traditional Chinese medicine for idiopathic pulmonary fibrosis (IPF). The aim of this study was to elucidate the influence of BHTF on induced IPF model through the aspect of histopathology and pulmonary function test. Wistar rats with bleomycin-induced IPF were given BHTF via intragastric gavage. After 14 days and 28 days of treatment, respectively, on these two time points, we first performed pulmonary function test, performed ventilation measure, and traced the Pressure-Volume Loop under anesthesia. Then, rats were sacrificed for hematoxylin-eosin and Masson's trichrome staining, immunohistochemistry staining of TGF-ß1 and α-SMA, and observation through transmission electron microscope. BHTF reduced infiltration of inflammation cells, collagen deposition, and fibrosis proliferation in pulmonary mesenchyme, inhibited the expression of TGF-ß1 and α-SMA, and avoided the abnormality of ultrastructure and quantities of lamellar bodies. It also ameliorated the parameters of FVC, MVV, PEF, FEF25, and Cdyn, maintained the shape of the Pressure-Volume Loop, and improved hysteresis. BHFT relieved the histopathologic changes, improved ventilation function, compliance, and work of breathing, meliorated the capacity and elasticity of the lungs, and stabilized the alveolar surface tension. Further speaking, it had a potential impact on the secretion of pulmonary surfactant.

Fruitless Wolfberry-Sprout Extract Rescued Cognitive Deficits and Attenuated Neuropathology in Alzheimer's Disease Transgenic Mice.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease featured by memory loss, neuroinflammation and oxidative stress. Overproduction or insufficient clearance of Aß leads to its pathological aggregation and deposition, which is considered the predominant neuropathological hallmark of AD. Therefore, reducing Aß levels and inhibiting Aß-induced neurotoxicity are feasible therapeutic strategies for AD treatment. Wolfberry has been traditionally used as a natural antioxidant and anti-aging product. However, whether wolfberry species has therapeutic potential on AD remains unknown. METHOD: The effects of fruitless wolfberry-sprout extract (FWE) on Aß fibrillation and fibril disaggregation was measured by thioflavin T fluorescence and transmission electron microscope imaging; Aß oligomer level was determined by dot-blot; Cell viability and apoptosis was assessed by MTT and TUNEL assay. The levels of Aß40/42, oxidative stress biomarkers and inflammatory cytokines were detected by corresponding kits. 8-month-old male APP/PS1 mice and their age-matched WT littermates were treated with FWE or vehicle by oral administration (gavage) once a day for 4 weeks. Then the cognitive performance was determined using object recognition test and Y-maze test. The Aß burden and gliosis was evaluated by immunostaining and immunoblotting, respectively. RESULTS: FWE significantly inhibited Aß fibrillation and disaggregated the formed Aß fibrils, lowered Aß oligomer level and Aß-induced neuro-cytotoxicity, and attenuated oxidative stress in vitro. Oral administration of FWE remarkably improved cognitive function, reduced Aß burden, decreased gliosis and inflammatory cytokines release, and ameliorated oxidative stress in the brains of APP/PS1 mice. CONCLUSION: These findings indicate that FWE is a promising natural agent for AD treatment.

Neural oscillations associated with auditory duration maintenance in working memory.

The neural representation of auditory duration remains unknown. Here, we used electroencephalogram (EEG) recordings to investigate neural oscillations during the maintenance of auditory duration in working memory (WM). EEG analyses indicated that the auditory duration length was not associated with changes in the theta band amplitude, whereas the alpha band amplitudes during 3-s and 4-s auditory duration conditions were lower than during the 1-s and 2-s conditions. Moreover, the alpha band amplitude and accuracy were positively correlated in the 2-s duration condition. We also found that the neural representation of auditory duration is segmented, with a critical threshold point of approximately 2 s, which is shorter than that for visual duration (3 s). The results emphasised the involvement of the alpha band in auditory duration maintenance in WM. Our study's findings indicate that different internal representations of auditory durations are maintained in WM below and above 2 s from the perspective of electrophysiology. Additionally, the critical threshold point is related to the sensory modality of duration.

Characterization of BcMF23a and BcMF23b, two putative pectin methylesterase genes related to pollen development in Brassica campestris ssp. chinensis.

Two homologous genes, Brassica campestris Male Fertility 23a (BcMF23a) and Brassica campestris Male Fertility 23b (BcMF23b), encoding putative pectin methylesterases (PMEs) were isolated from Brassica campestris ssp. chinensis (syn. Brassica rapa ssp. chinensis). These two genes sharing high sequence identity with each other were highly expressed in the fertile flower buds but silenced in the sterile ones of genic male sterile line system ('Bcajh97-01A/B'). Results of RT-PCR and in situ hybridization suggested that BcMF23a and BcMF23b were pollen-expressed genes, whose transcripts were first detected at the binucleate pollen and maintained throughout to the mature pollen grains. Western blot indicated that both of the putative BcMF23a and BcMF23b proteins are approximately 40 kDa, which exhibited extracellular localization revealed by transient expression analysis in the onion epidermal cells. The promoter of BcMF23a was active specifically in pollen during the late pollen developmental stages, while, in addition to the pollen, BcMF23b promoter drove an extra gene expression in the valve margins, abscission layer at the base of the first true leaves, taproot and lateral roots in seedlings.

Thrombin mutant W215A/E217A treatment improves neurological outcome and attenuates central nervous system damage in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination and axonal damage of the central nervous system. The pathogenesis of MS has also been linked to vascular inflammation and local activation of the coagulation system, resulting in perivascular fibrin deposition. Treatment of experimental autoimmune encephalomyelitis (EAE), a model of human MS, with antithrombotic and antiinflammatory activated protein C (APC) reduces disease severity. Since recombinant APC (Drotecogin alfa), originally approved for the treatment of severe sepsis, is not available for human MS studies, we tested the hypothesis that pharmacologic activation of endogenous protein C could likewise improve the outcome of EAE. Mice were immunized with murine myelin oligodendrocyte glycoprotein (MOG) peptides and at the onset of EAE symptoms, were treated every other day with either WE thrombin (25 µg/kg; i.v.), a selective recombinant protein C activator thrombin analog, or saline control. Mice were monitored for changes in disease score until euthanized for ex vivo analysis of inflammation. Administration of WE thrombin significantly ameliorated clinical severity of EAE, reduced inflammatory cell infiltration and demyelination, suppressed the activation of macrophages comprising the CD11b + population and reduced accumulation of fibrin (ogen) in the spinal cord. These data suggest that symptomatic MS may respond to a treatment strategy that involves temporal pharmacological enhancement of endogenous APC generation.

Activated scavenger receptor A promotes glial internalization of aß.

Beta-amyloid (Aß) aggregates have a pivotal role in pathological processing of Alzheimer's disease (AD). The clearance of Aß monomer or aggregates is a causal strategy for AD treatment. Microglia and astrocytes are the main macrophages that exert critical neuroprotective roles in the brain. They may effectively clear the toxic accumulation of Aß at the initial stage of AD, however, their functions are attenuated because of glial overactivation. In this study, we first showed that heptapeptide XD4 activates the class A scavenger receptor (SR-A) on the glia by increasing the binding of Aß to SR-A, thereby promoting glial phagocytosis of Aß oligomer in microglia and astrocytes and triggering intracellular mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, XD4 enhances the internalization of Aß monomers to microglia and astrocytes through macropinocytosis or SR-A-mediated phagocytosis. Furthermore, XD4 significantly inhibits Aß oligomer-induced cytotoxicity to glial cells and decreases the production of proinflammatory cytokines, such as TNF-α and IL-1ß, in vitro and in vivo. Our findings may provide a novel strategy for AD treatment by activating SR-A.

Causal interactions between the cerebral cortex and the autonomic nervous system.

Mental states such as stress and anxiety can cause heart disease. On the other hand, meditation can improve cardiac performance. In this study, the heart rate variability, directed transfer function and corrected conditional entropy were used to investigate the effects of mental tasks on cardiac performance, and the functional coupling between the cerebral cortex and the heart. When subjects tried to decrease their heart rate by volition, the sympathetic nervous system was inhibited and the heart rate decreased. When subjects tried to increase their heart rate by volition, the parasympathetic nervous system was inhibited and the sympathetic nervous system was stimulated, and the heart rate increased. When autonomic nervous system activity was regulated by mental tasks, the information flow from the post-central areas to the pre-central areas of the cerebral cortex increased, and there was greater coupling between the brain and the heart. Use of directed transfer function and corrected conditional entropy techniques enabled analysis of electroencephalographic recordings, and of the information flow causing functional coupling between the brain and the heart.

A novel HLA-DRα1-MOG-35-55 construct treats experimental stroke.

Chemoattraction of leukocytes into the brain after induction of middle cerebral artery occlusion (MCAO) increases the lesion size and worsens disease outcome. Our previous studies demonstrated that partial MHC class II constructs can reverse this process. However, the potential application of pMHC to human stroke is limited by the need to rapidly match recipient MHC class II with the ß1 domain of the pMHC construct. We designed a novel recombinant protein comprised of the HLA-DRα1 domain linked to MOG-35-55 peptide but lacking the ß1 domain found in pMHC and treated MCAO after 4 h reperfusion in humanized DR2 mice. Infarct volumes were quantified after 96 h reperfusion and immune cells from the periphery and CNS were evaluated for expression of CD74 and other cell surface, cytokine and pathway markers. This study demonstrates that four daily treatments with DRα1-MOG-35-55 reduced infarct size by 40 % in the cortex, striatum and hemisphere, inhibited the migration of activated CD11b+CD45high cells from the periphery to the brain and reversed splenic atrophy. Furthermore, DRα1-MOG-35-55 bound to CD74 on monocytes and blocked both binding and downstream signaling of macrophage migration inhibition factor (MIF) that may play a key role in infarct development. The novel DRα1-MOG-35-55 construct is highly therapeutic in experimental stroke and could be given to all patients at least 4 h after stroke onset without the need for tissue typing due to universal expression of DRα1 in humans.

Volitional control of the heart rate.

The heart rate is largely under control of the autonomic nervous system. The aim of the present study is to investigate the interactions between the brain and heart underlying volitional control of the heart and to explore the effectiveness of volition as a strategy to control the heart rate without biofeedback. Twenty seven healthy male subjects voluntarily participated in the study and were instructed to decrease and increase their heart beats according to rhythmic, computer generated sound either 10% faster or slower than the subjects' measured heart rate. Sympathetic and parasympathetic activities were estimated with the heart rate variability (HRV) obtained by power spectral analysis of RR intervals. Functional coupling patterns of cerebral cortex with the heart were determined by Partial directed coherence (PDC). In HR(slow) task; HR and sympathetic activity significantly decreased. However parasympathetic activity and power spectral density of EEG in low Alpha (8-10.5 Hz) band significantly increased. Moreover information flow from parietal area (P3 and P4) to RR interval significantly increased. During HR(quick) task; HR, sympathetic activity and power spectral density of EEG in low Beta (14-24 Hz) band significantly increased. Parasympathetic activity significantly decreased. Information flow from FT8, CZ and T8 electrodes to RR interval significantly increased. Our findings suggested that the heart beat can be controlled by volition and is related to some special areas in the cortex.

Sequence characterization and expression pattern of BcMF21, a novel gene related to pollen development in Brassica campestris ssp. chinensis.

In this report a full length cDNA, Brassica campestris Male Fertile 21 (BcMF21) was successfully isolated from one of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript-derived fragments (TDFs), BBP10, that was found down-regulated in the flower buds of sterile plants in Brassica campestris L. ssp. chinensis Makino genic male sterile (GMS) AB line system (Bcajh 97-01A/B). BcMF21 protein structure analysis showed a signal peptide at the N-terminus; two protein kinase C phosphorylation sites, five N-myristoylation sites and one casein kinase II phosphorylation site. The promoter region of BcMF21, a 779 bp upstream of ATG was isolated by thermal asymmetric interlaced-PCR (TAIL-PCR). Bioinformatics analysis revealed that the promoter of BcMF21 contained several classical cis-acting elements and three pollen specific elements. Transient expression analysis showed that the promoter could drive green fluorescence protein (GFP) expression. Quantitative reverse transcript-PCR analysis revealed that BcMF21 was specifically expressed in flower buds. The transcript level of BcMF21 was much lower in the sterile flower buds than in the fertile flower buds in 'Bcajh 97-01A/B' system. In situ hybridization further showed that BcMF21 was only expressed in the tetrads and the microspores at the tetrad stage and the uninucleate stage. In addition, phylogenetic analysis demonstrated that the BcMF21 was relative conserved within family Crucifereae and might be originated from the ancestor diploid B. campestris within genus Brassica according to the Triangle of U theory.

BcMF20, a putative pollen-specific transcription factor from Brassica campestris ssp. chinensis.

The Arabidopsis transcription factor (At1g26610) was shown to be down-regulated in male sterile lines compared to their maintainer line in Chinese cabbage (Brassica campestris ssp. chinensis) in our previous study. The BcMF20 gene, homologue of the At1g26610 gene was isolated from Chinese cabbage. It encodes a putative C2H2 zinc finger protein. The spatial and temporal expression patterns examined by qRT-PCR and in situ hybridization, indicated BcMF20 is specifically expressed in the developing pollen grains and the tapetum from the uninucleate pollen stage to mature pollen stage.

A polygalacturonase inhibitory protein gene (BcMF19) expressed during pollen development in Chinese cabbage-pak-choi.

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.

Analysis of genetic diversity in cytoplasmic male sterility, and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee).

In our previous study, we bred a stable cytoplasmic male sterility (CMS) line of tuber mustard by using distant hybridization and subsequent backcrosses. In this CMS plants, all floral organs are normal except the anthers, which are transformed into petals or tubular structures. Recently, 2 mitochondrial genes-atpA and orf220-that are distinctively present in the CMS line of tuber mustard were cloned and partially characterized. In our study of genetic diversity analysis of CMS, 7 species of Brassica and Raphanus crops, which included 5 CMS lines and their respective maintainer lines, were used to compare the constitution of protein-coding genes in the mitochondrial genomes. In 4 of the 43 mitochondrial genes, namely, atpA, orf220, orf256, and orf305/orf324, polymorphisms were detected among the tuber mustard CMS line and its maintainer line. The results of a cluster analysis indicate that petaloid CMS phenotype of tuber mustard is a novel CMS type and is nearer to the nap CMS in Brassica napus at the phylogenetic level. The results of individual amplifications of these genes indicate the presence of 4 sequence-characterized amplified region (SCAR) markers, which enable rapid and reliable identification of this CMS. Expressions of the orf220 and orf256 genes were detected only in the CMS line, while expression of the orf305 gene was detected in the maintainer line. The different expression patterns of different mitochondrial-specific marker genes indicate that the quantity of mitochondrial proteins is differentially regulated during organ/tissue development in tuber mustard. The results of this study suggest that the above mentioned 4 mitochondrial genes are associated with the petaloid CMS phenotype in tuber mustard.

Functional analysis of a pollen-expressed polygalacturonase gene BcMF6 in Chinese cabbage (Brassica campestris L. ssp. chinensis Makino).

In our earlier work, a pollen-expressed polygalacturonase gene BcMF6 was isolated from floral bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling and rapid amplification of cDNA ends (RACE). To unravel the biological function of BcMF6 gene, the antisense fragment from the BcMF6 gene with A9 promoter and CaMV35S promoter was transferred into flowering Chinese cabbage (B. campestris ssp. chinensis var. parachinensis). Out of transgenic plants transformed with the antisense gene constructed from the BcMF6, transgenic line with A9 promoter have a similar appearance to that with CaMV35S promoter. Morphological investigations showed that the transgenic plants developed the smaller floral organ with thin anther and less pollen. Pollen germination test indicated that only near 50% the pollen from the transgenic line could normally germinate. Further scanning electron microspore analysis of transgenic plants confirmed that half of pollen was abnormal. Cytological comparisons of microspore development also demonstrated that process of microsporogenesis was held up, microspores maturation was disrupted and pollen grain fail to separate, finally. In a word, the present study revealed that BcMF6, as a polygalacturonase gene, has a role in pollen maturation and pollen tube growth.

BcMF5, a pollen coat protein gene (PCP), from Brassica rapa. ssp. chinensis, involved in the transcription of different lengths of 3'-UTRs of PCPs.

Two transcript-derived fragments (GenBank accession number DN237907.1 and DN237908.1) with high homology accumulated in the wild-type flower buds of Chinese cabbage (Brassica rapa L. ssp. chinensis Makino) are isolated and investigated. By rapid amplification of cDNA ends (RACE), the full length cDNA of the two fragments were obtained. The alignment of their cDNA sequence showed that they are identical except for differences in a few nucleotides and should belong to the same gene, namely, B rassica rapa M ale F ertile 5 (BcMF5). The BcMF5 gene consists of 252 bp encoding a protein of 83 amino acids and is interrupted by an intron of 256 bp. Sequence blast analysis revealed that BcMF5 is a member of the pollen coat protein (PCP) gene family and shared a high homology to SLR-BP. In the process of 3'RACE, eight different lengths of 3'-UTR sequence are found from the wild type of the mmc mutant. Southern blot analysis showed that BcMF5 could be a single-copy gene in the Chinese cabbage genome, implying that eight different lengths of 3'-UTR sequences might come from the same gene and could be a result of multiple sites polyadenylation of 3'-UTRs of BcMF5. Based on sequence analysis, southern hybridization combined with RT-PCR, and northern hybridization, it was discovered that 3'-UTRs of BcMF5 contained some functional elements and their temporal and spatial expression patterns were different, but all strongly expressed in the stage IV and stage V flower buds of wild type. This indicate that different lengths of 3'-UTR may be involved in a regulation mechanism during the transcription of BcMF5.