A New Method for the Synthesis of 2′-O-Benzyladenosine Using Mitsunobu Reaction

Abstract

A new method to introduce a benzyl group onto the 2′-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3′-O-benzoylriboside and its 5′-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2′-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2′ is retained.     

Plasmid Transduction Using Bacteriophage Φadh for Expression of CC Chemokines by Lactobacillus gasseri ADH

Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Φadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Φadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.In sub-Saharan Africa, HIV infections are acquired predominantly via heterosexual contact and women are at greatest risk of being infected, accounting for 60% of HIV infections (27). Currently, there is no effective vaccine against HIV, and therefore, the development of topical microbicides for the prevention of viral entry at the cervicovaginal and rectal mucosal surfaces could serve as a potentially important alternative means of preventing HIV infection via vaginal and possibly rectal intercourse. To eliminate the requirement for precoital application of microbicides, an alternative live-microbicide strategy whereby nonpathogenic bacteria capable of colonizing the genital or gut mucosa are engineered to secrete HIV inhibitors has been investigated (13, 3, 20). Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, usually L. crispatus, L. jensenii, L. gasseri, and L. iners, which serve as an important natural barrier to HIV infection (1, 29, 17, 8). The colonization of the vaginal mucosa by engineered microbicide-secreting Lactobacillus strains would therefore potentially provide an economical and long-lasting method of enhancing this natural mucosal barrier. However, the persistence of such engineered strains within the vaginal mucosal milieu may require that these strains exhibit a selective advantage over endogenous bacterial populations, which could lead to potentially undesirable perturbations of the host''s existing microflora.We asked whether an alternative and potentially less disruptive strategy than the introduction of exogenous strains into the cervicovaginal mucosa could be utilized, whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via bacteriophage-mediated transfer of plasmid DNA (transduction). Transduction is a well-established phenomenon for a variety of bacterial species and has been used as a convenient and often more reliable method of DNA transfer than conventional methods (e.g., electroporation) for Gram-positive bacteria such as Staphylococcus species (4). Thus, the introduction of transducing phage particles specific for resident Lactobacillus species into the cervicovaginal milieu, subsequent transduction, and the secretion of antiviral molecules would negate the need for the introduction of exogenous engineered bacteria, which would have to compete with resident microflora.Few studies to date, however, have investigated transduction in Lactobacillus (26, 23, 21), and to our knowledge, there has been no attempt as yet to utilize transduction as a means of transferring expression plasmids into Lactobacillus species for the expression and secretion of anti-HIV molecules. Therefore, in this study, we undertook a proof-of-concept investigation to determine the feasibility of using transduction for this purpose. We show that high-frequency transduction into L. gasseri ADH by transducing particles derived from the Φadh phage can be achieved, where transducing particles contain pGK12-based expression plasmids (in concatemeric form) and transduction results in the production of transformants that express and secrete the CC chemokines and HIV coreceptor antagonists CCL5 (RANTES) and CCL3 (30).     

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.     

Development of a New Method to Get a Reliable Powder Flow Characteristics Using Only 1 to 2 g of Powder

In powder technology, it is often important to directly measure real powder flow rate from a small amount of powder. For example, in pharmaceutical industry, a frequent problem is to determine powder flow properties of new active pharmaceutical ingredient (API) in an early stage of the development when the amount of API is limited. The purpose of this paper is to introduce a new direct method to measure powder flow when the material is poorly flowing (cohesive) and the amount of material is about 1 to 2 g. The measuring system was simple, consisting of a flow chamber and electronic balance and an automated optical detection system, and for each measurement, only 1 to 2 g of sample was required. Based on the results obtained with this testing method, three selected sugar excipients, three grades of microcrystalline cellulose, and APIs (caffeine, carbamazepine, and paracetamol) can be classified as freely flowing, intermediate flowing, and poorly flowing powders, respectively. The average relative standard deviation for the flow time determinations was not more than 2–10%. The present novel flowability testing method provides a new tool for a rapid determination of flowing characteristics of powders (e.g., inhalation powders) and granules at a small scale.     

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: Use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4--glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43–59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37°C. No advantage of use was evident for the other GMOs.     

Measuring Adult Mortality Using Sibling Survival: A New Analytical Method and New Results for 44 Countries, 1974–2006

Background

For several decades, global public health efforts have focused on the development and application of disease control programs to improve child survival in developing populations. The need to reliably monitor the impact of such intervention programs in countries has led to significant advances in demographic methods and data sources, particularly with large-scale, cross-national survey programs such as the Demographic and Health Surveys (DHS). Although no comparable effort has been undertaken for adult mortality, the availability of large datasets with information on adult survival from censuses and household surveys offers an important opportunity to dramatically improve our knowledge about levels and trends in adult mortality in countries without good vital registration. To date, attempts to measure adult mortality from questions in censuses and surveys have generally led to implausibly low levels of adult mortality owing to biases inherent in survey data such as survival and recall bias. Recent methodological developments and the increasing availability of large surveys with information on sibling survival suggest that it may well be timely to reassess the pessimism that has prevailed around the use of sibling histories to measure adult mortality.

Methods and Findings

We present the Corrected Sibling Survival (CSS) method, which addresses both the survival and recall biases that have plagued the use of survey data to estimate adult mortality. Using logistic regression, our method directly estimates the probability of dying in a given country, by age, sex, and time period from sibling history data. The logistic regression framework borrows strength across surveys and time periods for the estimation of the age patterns of mortality, and facilitates the implementation of solutions for the underrepresentation of high-mortality families and recall bias. We apply the method to generate estimates of and trends in adult mortality, using the summary measure ₄₅ q ₁₅—the probability of a 15-y old dying before his or her 60th birthday—for 44 countries with DHS sibling survival data. Our findings suggest that levels of adult mortality prevailing in many developing countries are substantially higher than previously suggested by other analyses of sibling history data. Generally, our estimates show the risk of adult death between ages 15 and 60 y to be about 20%–35% for females and 25%–45% for males in sub-Saharan African populations largely unaffected by HIV. In countries of Southern Africa, where the HIV epidemic has been most pronounced, as many as eight out of ten men alive at age 15 y will be dead by age 60, as will six out of ten women. Adult mortality levels in populations of Asia and Latin America are generally lower than in Africa, particularly for women. The exceptions are Haiti and Cambodia, where mortality risks are comparable to many countries in Africa. In all other countries with data, the probability of dying between ages 15 and 60 y was typically around 10% for women and 20% for men, not much higher than the levels prevailing in several more developed countries.

Conclusions

Our results represent an expansion of direct knowledge of levels and trends in adult mortality in the developing world. The CSS method provides grounds for renewed optimism in collecting sibling survival data. We suggest that all nationally representative survey programs with adequate sample size ought to implement this critical module for tracking adult mortality in order to more reliably understand the levels and patterns of adult mortality, and how they are changing. Please see later in the article for the Editors'' Summary     

A New Efficient Stereoselective Method for the Synthesis of (E)-5-Aminoallyl-Pyrimidine-5′-Triphosphates Using Palladium-Catalyzed Heck Reaction

An efficient overall two-step strategy for the synthesis of (E)-5-aminoallyl-pyrimidine-5′-triphoshate, starting from commercially available pyrimidine-5′-triphosphate is described. The method involves regioselective iodination of pyrimidine-5′-triphosphate, followed by the palladium-catalyzed Heck coupling with allylamine. The catalytic reaction is highly stereoselective and compatible with many functional groups present in the reactants.     

RAPID ESTIMATION OF BLOOD SUGAR CONTENT—A New Method Using Clinitest? Tablets

A new, simple method for estimation of the sugar content of the blood, employing Clinitest® tablets such as are used for urine tests, gives quick information in cases of suspected diabetic acidosis.     

F?rster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method

Background

While the static structure of the intracellular Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1) has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes.

Methodology/Principal Findings

A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His) tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni²⁺/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP). GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm). Cy3NTA bound to His₁₀-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His₁₀ tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His₁₀ tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other.

Conclusions/Significance

These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1 structure.     

A Convenient Method for the Synthesis of 2-(β-D-Glycopyranosylthio) Pyridines

Abstract

Treatment of piperidinium salts of dihydropyridinethiolates 3 with glycosyl bromides 4 in dry acetone provides a convenient and high yielding synthesis of 1,4-dihydro-3-cyanopyridine thioglycosides 5. The structures of 5 were confirmed by oxidation as well as by ¹H NMR and ¹³C NMR spectral analysis.     

Development and Validation of a New Method to Measure Walking Speed in Free-Living Environments Using the Actibelt? Platform

Walking speed is a fundamental indicator for human well-being. In a clinical setting, walking speed is typically measured by means of walking tests using different protocols. However, walking speed obtained in this way is unlikely to be representative of the conditions in a free-living environment. Recently, mobile accelerometry has opened up the possibility to extract walking speed from long-time observations in free-living individuals, but the validity of these measurements needs to be determined. In this investigation, we have developed algorithms for walking speed prediction based on 3D accelerometry data (actibelt®) and created a framework using a standardized data set with gold standard annotations to facilitate the validation and comparison of these algorithms. For this purpose 17 healthy subjects operated a newly developed mobile gold standard while walking/running on an indoor track. Subsequently, the validity of 12 candidate algorithms for walking speed prediction ranging from well-known simple approaches like combining step length with frequency to more sophisticated algorithms such as linear and non-linear models was assessed using statistical measures. As a result, a novel algorithm employing support vector regression was found to perform best with a concordance correlation coefficient of 0.93 (95%CI 0.92–0.94) and a coverage probability CP1 of 0.46 (95%CI 0.12–0.70) for a deviation of 0.1 m/s (CP2 0.78, CP3 0.94) when compared to the mobile gold standard while walking indoors. A smaller outdoor experiment confirmed those results with even better coverage probability. We conclude that walking speed thus obtained has the potential to help establish walking speed in free-living environments as a patient-oriented outcome measure.     

A New Solid Phase Method for the Synthesis of Oligonucleotides with Terminal -3′-Phosphate

Abstract

A novel method for the synthesis of oligonucleotides with terminal-3′-phosphate using universal CPG polymer support is described.     

A Novel Method Using 8-hydroperoxy-2′-deoxyguanosine Formation for Evaluating Antioxidative Potency

Degenerative diseases such as cancer are induced by oxidative genetic damage. Antioxidants can scavenge reactive oxygen species, but to prevent disease, they must do so quickly, before the DNA bases are damaged. In the present study, a novel method was established for evaluating the potency of antioxidants employing 2'-deoxyguanosine as a target and 2,2'-azobis(2-amidinopropane) dihydrochloride as a reactive oxygen generator. The reaction formed one product linearly with time. This product was a novel 8-hydroperoxy-2'-deoxyguanosine (8-OOHdG). Using this system, 81 antioxidants occurring in our diet were assayed for activity to suppress the formation of 8-OOHdG by high-performance liquid chromatography (HPLC). The system was useful for the evaluation of antioxidative potency, compared to another method utilizing 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further, it was enabled to examine the synergism of antioxidants. The formation of 8-OOHdG started only after the antioxidants had been consumed. Ascorbic acid, quercetin, and epigallocatechin gallate together delayed the formation by the sum total of the delay times of each factor alone. The proposed method is simple and easy, and can evaluate which dietary antioxidants inhibit reactive oxygen species more quickly than the DNA bases are damaged.     

Binding of longer Aβ to transmembrane domain 1 of presenilin 1 impacts on Aβ42 generation

Background

Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.

Results

We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.

Conclusions

Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production.