Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer ...snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus ...greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species ...during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.
Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only ...advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.
A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.
These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Podophyllum species are sources of (−)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, ...however, remains largely unknown, with the last unequivocally demonstrated intermediate being (−)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (−)-matairesinol into (−)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (−)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.
Background: Biosynthetic pathways to structurally complex plant medicinals are incomplete or unknown.
Results: Next generation sequencing/bioinformatics and metabolomics analysis of Podophyllum tissues gave putative unknown genes in podophyllotoxin biosynthesis.
Conclusion: Regio-specific methylenedioxy bridge-forming CyP450s were identified catalyzing pluviatolide formation.
Significance: Database of several medicinal plant transcriptome assemblies and metabolic profiling are made available for scientific community.
Three-dimensional chromatin loop structures connect regulatory elements to their target genes in regions known as anchors. In complex plant genomes, such as maize, it has been proposed that loops ...span heterochromatic regions marked by higher repeat content, but little is known on their spatial organization and genome-wide occurrence in relation to transcriptional activity.
Here, ultra-deep Hi-C sequencing of maize B73 leaf tissue was combined with gene expression and open chromatin sequencing for chromatin loop discovery and correlation with hierarchical topologically-associating domains (TADs) and transcriptional activity. A majority of all anchors are shared between multiple loops from previous public maize high-resolution interactome datasets, suggesting a highly dynamic environment, with a conserved set of anchors involved in multiple interaction networks. Chromatin loop interiors are marked by higher repeat contents than the anchors flanking them. A small fraction of high-resolution interaction anchors, fully embedded in larger chromatin loops, co-locate with active genes and putative protein-binding sites. Combinatorial analyses indicate that all anchors studied here co-locate with at least 81.5% of expressed genes and 74% of open chromatin regions. Approximately 38% of all Hi-C chromatin loops are fully embedded within hierarchical TAD-like domains, while the remaining ones share anchors with domain boundaries or with distinct domains. Those various loop types exhibit specific patterns of overlap for open chromatin regions and expressed genes, but no apparent pattern of gene expression. In addition, up to 63% of all unique variants derived from a prior public maize eQTL dataset overlap with Hi-C loop anchors. Anchor annotation suggests that < 7% of all loops detected here are potentially devoid of any genes or regulatory elements. The overall organization of chromatin loop anchors in the maize genome suggest a loop modeling system hypothesized to resemble phase separation of repeat-rich regions.
Sets of conserved chromatin loop anchors mapping to hierarchical domains contains core structural components of the gene expression machinery in maize. The data presented here will be a useful reference to further investigate their function in regard to the formation of transcriptional complexes and the regulation of transcriptional activity in the maize genome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aim
The prevalence of obesity continues to rise, as does that of weakness. However, it is unclear how this impacts the risk of falling. We aimed to ascertain the risk of falls using new definitions ...of clinically defined weakness.
Methods
We applied clinically defined weakness definitions to the National Health and Aging Trends Survey using the Sarcopenia Definitions Outcomes Consortium cutpoints. Three exposure variables were created: grip‐strength‐defined weakness and body mass index GS/BMI‐defined obesity; weakness and obesity, weakness and waist circumference‐derived obesity (GS/WC); and weakness defined by a ratio of GS÷BMI. Proportional hazards modeled incident falls as a function of weakness with/without obesity (hazard ratio HR 95% confidence intervals).
Results
Of 4906 respondents aged ≥ 65 years (54.5% female), the mean ± SD grip strength, BMI, and WC were 26.7 ± 10.6 kg, 27.4 ± 5.4 kg/m2, and 99.5 ± 16.3 cm, respectively. Using the neither weakness/obesity as the referent, weakness was associated with incident falls across all definitions (GS/BMI: HR 1.19 1.07, 1.33; GS/WC: HR 1.39 1.19, 1.62; GS ÷ BMI: HR 1.16 1.05, 1.28). Weakness with obesity was associated with falls using GS/WC (HR 1.28 1.11, 1.48). Obesity status was associated with falls in both the BMI and the WC definition (1.17 1.02–1.35, 1.16 1.05–1.28).
Conclusion
Our findings further evaluate the definitions of clinically defined weakness with and without obesity in older adults. As falls are an important patient outcome, establishing this relationship is critical for both clinicians and researchers. Future study should identify high‐risk individuals in order to direct specific interventions to them. Geriatr Gerontol Int 2023; 23: 213–220.
We applied three of the Sarcopenia Definitions Outcomes Consortium definitions for clinically defined weakness to the National Health and Aging Trends Survey. Of 4906 respondents aged ≥ 65 years (54.5% female), using the neither weakness/obesity as the referent, weakness was associated with incident falls across all definitions (Grip Strength ÷ Body mass index: HR 1.19 1.07, 1.33; Grip Strength ÷ Waist Circumference: HR 1.39 1.19, 1.62; Grip Strength ÷ BMI: HR 1.16 1.05, 1.28). Findings further evaluate the definitions of clinically defined weakness with and without obesity in older adults.
Objectives
To determine the relationship between frailty and overall and cardiovascular mortality.
Design
Longitudinal mortality analysis.
Setting
National Health and Nutrition Examination Survey ...(NHANES) 1999–2004.
Participants
Community‐dwelling older adults aged 60 and older (N = 4,984; mean age 71.1 ± 0.19, 56% female).
Measurements
We used data from 1999–2004 cross‐sectional NHANES and mortality data from the National Death Index, updated through December 2011. An adapted version of Fried's frailty criteria was used (low body mass index, slow walking speed, weakness, exhaustion, low physical activity). Frailty was defined as persons meeting 3 or more criteria, prefrailty as meeting 1 or 2 criteria, and robust (reference) as not meeting any criteria. The primary outcome was to evaluate the association between frailty and overall and cardiovascular mortality. Cox proportional hazard models were used to evaluate the association between risk of death and frailty category adjusted for age, sex, race, smoking, education, coronary artery disease, heart failure, nonskin cancer, diabetes, and arthritis.
Results
Half (50.4%) of participants were classified as robust, 40.3% as prefrail, and 9.2% as frail. Fully adjusted models demonstrated that prefrail (hazard ratio (HR) = 1.64, 95% confidence interval (CI) = 1.45–1.85) and frail (HR = 2.79, 95% CI = 2.35–3.30) participants had a greater risk of death and of cardiovascular death (prefrail: HR = 1.84, 95% CI = 1.45–2.34; frail: HR = 3.39, 95% CI = 2.45–4.70).
Conclusion
Frailty and prefrailty are associated with increased risk of death. Demonstrating the association between prefrail status and mortality is the first step to identifying potential targets of intervention in future studies.
Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary ...plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These ...reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC$_{50}$ = 10 $\mu $M) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.
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