Studies on Nucleotide Chemistry 15. Synthesis of Oligodeoxynucleotides Using Amidine Protected Nucleosides

Abstract

An in situ method is described for synthesizing DNA which incorporates a new series of amidine protected deoxy-nucleosides and bis-dialkylaminophosphines as phosphitylating agents. These procedures were used to synthesize d(GGGAATTCCC) which was digested by EcoRI.     

Calmodulin Acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons

Summary Lateral axons from the abdominal nerve cord of cray-fish were internally perfused with the calcium receptor calmodulin (CaM) in solutions with low (pCa>7.0) or high (pCa 5.5) calcium concentrations and studied electrophysiologically and morphologically. Results from these experiments show that when the internal solution contains calcium-activated calmodulin (Ca²⁺-CaM) the junctional resistance between the axons increases from control values of about 60 to 500–600 k in 60 min. In contrast, axons perfused with calmodulin in low calcium solutions maintain their junctional resistance at control levels during the 60-min perfusion. Similar results are obtained when only one or both coupled axons are perfused.The morphological study shows that in the perfused axons the axoplasmic organelles are replaced or grossly perturbed by the perfusion solution up to the region of the synapses. Additionally, in axons perfused with Ca²⁺-CaM there are regions where the synaptic gap between the membranes decreases from a control 4–6 to 2–3 nm. Both electrophysiological and morphological results can be interpreted as indicating that calcium-activated calmodulin acts directly on the junctional channels to induce their closure.     

Influence of phytoplankton composition and stratification degree on gut pigment content of Ceriodaphnia sp. at dawn

Very short-term feeding activity of the cladoceran Ceriodaphniasp. was investigated in situ in a eutrophic reservoir in thesouth of Spain, using fluorimetric analysis of the gut pigmentcontent in periods when the water column was relatively mixedor strongly stratified. The results obtained in the mixed watercolumn showed a clear increase in gut pigment content at dawn,a period sampled with high frequency. The accumulation of thecladoceran at the depth of maximum concentration of phytoplankton,and the high gut pigment concentration in cladocerans at thatdepth just after dawn, suggested active feeding of Ceriodaphniaon phytoplankton at that time. During stratification, the abundanceof Ceriodaphnia was higher, but the gut pigment contents werevery low and they did not reflect any clear feeding patterns,with either time or depth. Changes in phytoplankton concentrationand composition between the relatively mixed and the stratifiedwater column suggest a shift in feeding activity from herbivorousto.     

Developmental changes in the breakdown of brain tubulin by cerebral cathepsin D

The activity of cathepsin D on hemoglobin and on cytoplasmic tubulin was measured in brain preparations at different ages—in newborn, 10-and 21-day-old, and young adult rats. Enzyme activity increased after birth, reaching a maximum at around 21 days, and then declined. This increase was not parallel with decreased turnover of proteins during development, but was parallel with decreasing level and increasing microheterogeneity and rate of assembly of tubulin during development. The breakdown of tubulin was heterogeneous, with initial fast breakdown of a large portion, followed by breakdown at a lower rate. This heterogeneity in breakdown persisted throughout development. The breakdown of tubulin, unlike that of hemoglobin, was at all ages greater at pH 5.8 than at pH 3.2. The possible role of cathepsin D in tubulin metabolism and the developmental changes under physiological conditions need further exploration.     

Analysis of mesophyll conductance in five understory herbaceous species

Mesophyll conductance (gm) has received over time much less attention than stomatal conductance (gs), although it affects leaf photosynthesis to about the same extent as stomatal conductance does. The objective of this study was to analyze the gm trend in five understory herbaceous species growing in a close-canopy forest in the north-west of Italy. In particular, three of analyzed species were monocots: Carex brizoides Lam., Carex pilosa Scop., and Oplismenus undulatifolius P. Beauv and the others dicots species: Circaea lutetiana L., and Pulmonaria officinalis Ced. The results showed, on one hand, the absence of correlation between gm and the considered environmental variables in the forest understory (i.e. air temperature, photosynthetic photon flux density and carbon dioxide concentration). Moreover, we carried out a principal component analysis considering all the analyzed morphological and physiological variables for the five species. The following correlation between the first component, related to the leaf mass per unit of leaf area and the leaf tissue density, and gm seem to suggest a key role of the leaf structural features in determining gm variations across the five species.     

Pelagic denitrification and methane oxidation in oxygen-depleted waters of the Louisiana shelf

Anthropogenic nutrient inputs fuel eutrophication and hypoxia ([O₂]?<?2 mg L^?1), threatening coastal and near shore environments across the globe. The world’s second largest anthropogenic coastal hypoxic zone occurs annually along the Louisiana (LA) shelf. Springtime loading of dissolved inorganic nitrogen (DIN) from the Mississippi River, combined with summertime stratification and increased water residence time on the shelf, promotes establishment of an extensive hypoxic zone that persists throughout the summer. We investigated the patterns of pelagic denitrification and methane (CH₄) oxidation along the LA shelf. Microbial activity rates were determined along with concentrations of dissolved nutrients and greenhouse gases, nitrous oxide (N₂O) and CH₄, during summer in 2013, 2015, and 2016. We documented denitrification rates up to 1900 nmol N L^?1 d^?1 and CH₄ oxidation rates as high as 192 nmol L^?1 d^?1 in hypoxic waters characterized by high concentrations of N₂O (range: 1 to 102 nM) and CH₄ (range: 3 to 641 nM). Ecosystem scaling estimates suggest that pelagic denitrification could remove between 0.1 and 47% of the DIN input from the Mississippi River, whereas CH₄ oxidation does not function as an effective removal process with CH₄ escaping to the atmosphere. Denitrification and CH₄ oxidizing bacteria within the LA shelf hypoxic zone were largely unable to keep up with the DIN and CH₄ inputs to the water column. Rates were variable and physiochemical dynamics appeared to regulate the microbial removal capacity for both nitrate/nitrite and CH₄ in this ecosystem.

     

Dynamic modulation of FGFR1–5-HT1A heteroreceptor complexes. Agonist treatment enhances participation of FGFR1 and 5-HT1A homodimers and recruitment of β-arrestin2

New findings show that neurotrophic and antidepressant effects of 5-HT in brain can, in part, be mediated by activation of the 5-HT1A receptor protomer in the hippocampal and raphe FGFR1–5-HT1A heteroreceptor complexes enhancing the FGFR1 signaling. The dynamic agonist modulation of the FGFR1–5-HT1A heteroreceptor complexes and their recruitment of β-arrestin is now determined in cellular models with focus on its impact on 5-HT1AR and FGFR1 homodimerization in the heteroreceptor complexes based on BRET² assays. The findings show that coagonist treatment with 8-OH-DPAT and FGF2 but not treatment with the 5-HT1A agonist alone markedly increases the BRETmax values and significantly reduces the BRET50 values of 5HT1A homodimerization. The effects of FGF2 or FGF20 with or without the 5-HT1A agonist were also studied on the FGFR1 homodimerization of the heteroreceptor complexes. FGF2 produced a marked and rapid increase in FGFR1 homodimerization which partially declined over a 10 min period. Cotreatment with FGF2 and 5-HT1A agonist blocked this decline in FGFR1 homodimerization. Furthermore, FGF2 alone produced a small increase in the BRET² signal from the 5-HT1A-β-arrestin2 receptor–protein complex which was additive to the marked effect of 8-OH-DPAT alone. Taken together, the participation of 5-HT1A and FGFR1 homodimers and recruitment of β-arrestin2 was demonstrated in the FGFR1–5-HT1A heteroreceptor complexes upon agonist treatments.     

27-Nor-Δ4-dafachronic acid is a synthetic ligand of Caenorhabditis elegans DAF-12 receptor

27-Nor-Δ⁴-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20β-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ⁴-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor.