A tobacco homolog of DCN1 is involved in pollen development and embryogenesis

Key Message

We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth.

Abstract

Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called DEFECTIVE IN CULLIN NEDDYLATION 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.     

The Effects of an Animal-Assisted Intervention on Salivary Alpha-Amylase,Salivary Immunoglobulin A,and Heart Rate during Forensic Interviews in Child Sexual Abuse Cases

Animal-assisted invention (AAI) in gaining attention as a therapeutic modality; however, the effect of it has not been well studied in the child welfare system. The purpose of this study was to examine the effects of AAI on stress indicators (as measured by salivary alpha-amylase (sAA), immunoglobulin A (IgA), and heart rate) in children undergoing forensic interviews for alleged sexual abuse. A repeated measures design was used in this study. Upon receiving signed, informed consents and assents, children were enrolled in the study. Children (n = 42), whose ages ranged from 5 to 14 years (M = 8.91, SD = 2.33), were assigned to either the intervention condition (n = 19; AAI during forensic interview) or the control condition (n = 23; standard practice forensic interview). Each child's parent/guardian completed a demographic form, and saliva samples and heart rate measures were obtained from each child before and after the forensic interview. Mixed linear models were tested, with the level of significance set at p ≤ 0.05. There was an interactive effect regarding the duration of the interview and the presence of the dog on sAA after the forensic interview (p = 0.047). There also was a significant interaction between age and length of interview (p = 0.01). Salivary immunoglobulin A tended to be lower (p = 0.055) when the therapy dog was present during the forensic interview. Results further indicated that the drop in heart rate was greater in longer interviews and with older children (p = 0.02) when the dog was present. Individuals working in child welfare systems can use the results of this study to advocate for the use of therapy dogs as a therapeutic intervention. More research is needed to further examine the relationships among AAI, salivary biomarkers, and stress responses in children to improve child welfare.     

Co-localized or randomly distributed? Pair cross correlation of in vivo grown subgingival biofilm bacteria quantified by digital image analysis

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 μm) with a pronounced peak at 1.5 μm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.     

The evolution of public review and oversight mechanisms in human gene transfer research: joint roles of the FDA and NIH

The federal government is critically examining its responsibilities and opportunities for bringing the new field of gene therapy to fruition and for assuring public confidence in this new area of biomedicine. The evolving mechanisms for review and regulation in human gene transfer studies in the United States are being enhanced by increasingly effective interactions between the Food and Drug Administration and the National Institutes of Health.     

Biogeochemical features of lipids in endolithic microbial communities in the Ross Desert (McMurdo Dry Valleys), Antarctica.

Endolithic microbial communities inhabiting porous rocks in the cold, dry mountainous regions of Antarctica have been studied extensively as examples of life's adaptations to extreme environments. Here, we examine hydrocarbons and fatty acids occurring in these communities in order to clarify their biogeochemical features with respect to source organisms, microbial activity, fossilization processes and the influence of Gondwanaland sediments. Unusually, long-chain (>C19) n-alkanes and anteiso-alkanes were often the major hydrocarbons in the samples. A suite of n-alkanoic acids (n-C9-n-C32) and long-chain anteiso-alkanoic acids (a-C20-a-C30) were found, along with short-chain iso- and anteiso-alkanoic acids, and n-alkenoic acids. The relationship between long-chain n-alkanoic acids (n-C20-n-C32) and long-chain anteiso-alkanoic acids suggests that these compounds probably originated from the same group of microorganisms, such as bacteria or endolithic lichens, under moderate pH conditions (pH 3-5). Relatively high trans/cis-C16:1 alkenoic acid ratios suggest the presence of unfavorable environmental conditions in the endolithic microbial habitat. Normal-alkenoic/alkanoic acid ratios may be a useful marker for the fossilization of endolithic microbial communities. Thermally matured triterpanes and steranes from fossilized associations on Mount Fleming strongly suggest the presence of Gondwanaland sediments formed during Devonian and Jurassic (400-180 million years ago).     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Persistent and acute central nervous system infections are caused by Theiler''s murine encephalomyelitis viruses which differ in RNA composition but code for only slightly different proteins

The RNA and proteins for four representatives of the two subgroups of Theiler's murine encephalomyelitis viruses were studied. The large RNase T1-resistant oligonucleotides, when mapped along the RNA molecules, were found to be differently distributed in the two subgroups. Replicative form RNAs of two representatives were partially denatured, and the denaturation maps obtained were found to be similar but not identical. In addition, the analysis of the tryptic maps of the capsid proteins of all four isolates revealed that only small differences in the peptide map patterns exist among these viruses. The correlation of these findings with the pathogenicity of Theiler's viruses is discussed.     

Swimming behavior of X and Y human sperm

Abstract. A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.     

Evidence for vasopressin production in the human gastrointestinal system.

In the present study we performed immunohistochemical examination of segments from the human gastrointestinal system for the presence of cells containing vasopressin (VP) and vasopressin-associated human neurophysin (VP-HNP). VP immunoreactivity was found in crypt cells of the stomach and small intestine, and in mononuclear cells within the lamina propria and submucosa. VP-HNP was demonstrated in the crypt and lamina propria regions of the small intestine, and was colocalized with vasopressin in crypt cells. This colocalization indicates local vasopressin synthesis by these cells and raises the possibility that they may perform an endocrine or exocrine function in the human gastrointestinal system.