A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues

Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphos-phates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (>90 000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wavelengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was .99% and replicate extractions within or between samples were highly reproducible (sd<5%). The single step method described minimizes potential losses associated with post-extraction manipulation and provides the capability to examine alterations in nucleotide precursor–product metabolism under various physiological and pharmacological conditions.     

Cyclooxygenase and 5-lipoxygenase inhibitory activity of 2,6 di-t-butylphenols linked by a sulfur atom to 1,3,4-thiadiazoles and 1,3,4-oxadiazoles

The preparation of a series of 1,3,4-thiadiazoles and 1,3,4-oxadizoles linked by a thioether to 2,6-di-t-butylphenol and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is dicussed.     

Anticonvulsant sugar sulfamates. Potent cyclic sulfate and cyclic sulfite analogues of topiramate

Exploration structure-activity relationships surrounding the clinically effective antiepileptic drug topiramate (1) led to a series of potent anticonvulsants with a 4,5-cyclic sulfate or 4,5-cycli sulfite functionality. Key derivative 2 (RWJ-37947) is ca. 8 times more potent than topiramate in mice; it also features a long duration of action and a very favorable neurotoxicity index.     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Correlation between arrested secondary plasmodesmal development and onset of accelerated leaf senescence in yeast acid invertase transgenic tobacco plants

Mature leaves of a transgenic tobacco plant ( Nicotiana tabacum L var. Samsun, line A41-10) that constitutively express a yeast-derived acid invertase gene develop symptoms which are characterized by the presence of greenish-yellow and green sectors in the same leaf, and onset of early, leaf senescence. Previous studies indicated that invertase activity was two- to threefold higher in the greenish-yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish-yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase-expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.     

Evaluating treatment effects in group sequential multivariate longitudinal studies with covariate adjustment

Jeffries et al. (2018) investigated testing for a treatment difference in the setting of a randomized clinical trial with a single outcome measured longitudinally over a series of common follow-up times while adjusting for covariates. That paper examined the null hypothesis of no difference at any follow-up time versus the alternative of a difference for at least one follow-up time. We extend those results here by considering multivariate outcome measurements, where each individual outcome is examined at common follow-up times. We consider the case where there is interest in first testing for a treatment difference in a global function of the outcomes (e.g., weighted average or sum) with subsequent interest in examining the individual outcomes, should the global function show a treatment difference. Testing is conducted for each follow-up time and may be performed in the setting of a group sequential trial. Testing procedures are developed to determine follow-up times for which a global treatment difference exists and which individual combinations of outcome and follow-up time show evidence of a difference while controlling for multiplicity in outcomes, follow-up, and interim analyses. These approaches are examined in a study evaluating the effects of tissue plasminogen activator on longitudinally obtained stroke severity measurements.     

Upper thermal limits predict herpetofaunal responses to forest edge and cover

Amphibians and reptiles are sensitive to changes in the thermal environment, which varies considerably in human-modified landscapes. Although it is known that thermal traits of species influence their distribution in modified landscapes, how herpetofauna respond specifically to shifts in ambient temperature along forest edges remains unclear. This may be because most studies focus on local-scale metrics of edge exposure, which only account for a single edge or habitat patch. We predicted that accounting for the combined effect of multiple habitat edges in a landscape would best explain herpetofaunal response to thermally mediated edge effects. We (1) surveyed herpetofauna at two lowland, fragmented forest sites in central Colombia, (2) measured the critical thermal maximum (CT_max) of the species sampled, (3) measured their edge exposure at both local and landscape scales, and (4) created a thermal profile of the landscape itself. We found that species with low CT_max occurred both further from forest edges and in areas of denser vegetation, but were unaffected by the landscape-scale configuration of habitat edges. Variation in the thermal landscape was driven primarily by changes in vegetation density. Our results suggest that amphibians and reptiles with low CT_max are limited by both canopy gaps and proximity to edge, making them especially vulnerable to human modification of tropical forest. Abstract in Spanish is available with online material.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.     

Coherent modeling of longitudinal causal effects on binary outcomes

Analyses of biomedical studies often necessitate modeling longitudinal causal effects. The current focus on personalized medicine and effect heterogeneity makes this task even more challenging. Toward this end, structural nested mean models (SNMMs) are fundamental tools for studying heterogeneous treatment effects in longitudinal studies. However, when outcomes are binary, current methods for estimating multiplicative and additive SNMM parameters suffer from variation dependence between the causal parameters and the noncausal nuisance parameters. This leads to a series of difficulties in interpretation, estimation, and computation. These difficulties have hindered the uptake of SNMMs in biomedical practice, where binary outcomes are very common. We solve the variation dependence problem for the binary multiplicative SNMM via a reparameterization of the noncausal nuisance parameters. Our novel nuisance parameters are variation independent of the causal parameters, and hence allow for coherent modeling of heterogeneous effects from longitudinal studies with binary outcomes. Our parameterization also provides a key building block for flexible doubly robust estimation of the causal parameters. Along the way, we prove that an additive SNMM with binary outcomes does not admit a variation independent parameterization, thereby justifying the restriction to multiplicative SNMMs.