Proteomic associations with forced expiratory volume - a Mendelian randomisation study.

A decline in forced expiratory volume (FEV1) is a hallmark of obstructive respiratory diseases, an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1. Data from the general population-based AGES-Reykjavik study were used. Proteomic measurements were done using 4,782 DNA aptamers (SOMAmers). Data from 1,648 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5,368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102). In observational analyses, 473 SOMAmers were associated with FEV1 after multiple testing adjustment. The most significant were R-Spondin 4, Alkaline Phosphatase, Placental Like 2 and Retinoic Acid Receptor Responder 2. Of the 235 SOMAmers with genetic data, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta and Apolipoprotein M. THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments. In summary, this large scale proteogenomic analyses of FEV1 reveals protein markers of FEV1, as well as several proteins with potential causality to lung function.

Ligand regulation of juvenile hormone binding protein mRNA in mutant Manduca sexta.

Insect hemolymph juvenile hormone binding protein (hJHBP) regulates peripheral titers of its ligands, the juvenile hormones. In larvae of the black (bl) strain of the tobacco hornworm, Manduca sexta, treatment with small doses of juvenile hormone I (JH I) can also regulate titers of hJHBP. To further investigate this regulation, responsiveness of hJHBP mRNA expression to JH I was characterized in vivo. RNA analyzes revealed that transcript levels in fat body, the site of hJHBP synthesis, increased fivefold within several hours of treatment with physiological doses of hormone and remained elevated for approximately 16 h. Sensitivity to JH treatment was found to vary temporally. To ensure transcript identity, a wild-type cDNA clone and a bl RT-PCR fragment were sequenced and found to be 99% homologous. Together, these results suggest that JH participates in regulating expression of its transport protein in bl larvae by modifying the in vivo abundance of hJHBP's mRNA transcript.

Sequence, structure and expression of the hemolymph juvenile hormone binding protein gene in the tobacco hornworm, Manduca sexta.

The hemolymph juvenile hormone binding protein (hJHBP) gene of Manduca sexta is a key target of its specific ligand, juvenile hormone (JH). While the cDNA for hJHBP has been partially characterized, little is known about the hJHBP gene structure or its promoter(s) and enhancers(s). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. To better understand the underlying molecular events affecting regulation, we sequenced the M. sexta hJHBP gene and its mRNA transcript, characterized its genomic organization, and determined the spatial and temporal expression patterns of the hJHBP gene. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. The earliest expression of hJHBP occurs 24 to 48 h after fertilization. Distribution studies indicate that fat body is the only site for hJHBP expression. Elements displaying similarity with sequences of other lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements.

The lethal effects of Cyperus iria on Aedes aegypti.

The sedge Cyperus iria, a common weed in rice, contains large amounts of the insect hormone (10R) juvenile hormone III (JH III). Given its widespread distribution in Asia and Africa, we examined the possibility that C. iria could be used as a safe, inexpensive, and readily available mosquito larvicide. Plants of varying ages were harvested and leaves tested for lethal effects on larvae of the yellow fever mosquito, Aedes aegypti. The median lethal doses (LD50s) for frozen leaves from 1- and 2-month-old plants were 267 and 427 mg/100 ml of water, respectively. Leaves from 1-month-old C. iria contained 193 micrograms JH III/g fresh weight, whereas leaves from 2-month-old plants contained 143 micrograms JH III/g fresh weight. Larval sensitivity to the plant differed with age; 4-day-old larvae displayed the greatest mortality followed in decreasing sensitivity by larvae 5, 6, 3, and 2 days old. Six Cyperus species (C. albostriatus, C. alternifolius, C. esculentus, C. iria, C. miliifolius, and C. papyrus) of similar developmental stage were assayed for JH III content. Only C. iria was found to contain significant levels of JH III.

A strategy for probing the function of noncoding RNAs finds a repressor of NFAT.

Noncoding RNA molecules (ncRNAs) have been implicated in numerous biological processes including transcriptional regulation and the modulation of protein function. Yet, in spite of the apparent abundance of ncRNA, little is known about the biological role of the projected thousands of ncRNA genes present in the human genome. To facilitate functional analysis of these RNAs, we have created an arrayed library of short hairpin RNAs (shRNAs) directed against 512 evolutionarily conserved putative ncRNAs and, via cell-based assays, we have begun to determine their roles in cellular pathways. Using this system, we have identified an ncRNA repressor of the nuclear factor of activated T cells (NFAT), which interacts with multiple proteins including members of the importin-beta superfamily and likely functions as a specific regulator of NFAT nuclear trafficking.

Recent advances in radioimmunoassay technology for the juvenile hormones.

Recent refinements in juvenile hormone radioimmunoassay technology now make this method significantly more sensitive and easier to use. Rabbit polyclonal antisera against (10R) JH III and racemic JH II have been developed to determine hemolymph hormone titers in the low picogram range. The antisera display minimal cross-reactivity with JH metabolites, JH analogs, and hemolymph lipids. One antiserum recognizes racemic JH I, II, and (10R) III almost equivalently, exhibiting 50% displacement between 100 and 130 pg per tube. Another antiserum is JH II-specific and exhibits 50% displacement at 35 pg per tube. Assay sensitivity has been enhanced by using (10R,11S) [methyl-3H]-JH II of very high specific activity (> 80 Ci/mmol) generated with Hyalophora cecropia accessory gland S-adenosylmethionine transferase and S-[methyl-3H]-adenosyl-L-methionine. Preparation of biological samples has been simplified with overall recoveries of JH from hemolymph ranging between 60 and 75%.