Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

Secukinumab provides sustained PASDAS-defined remission in psoriatic arthritis and improves health-related quality of life in patients achieving remission: 2-year results from the phase III FUTURE 2 study

Background

Secukinumab has demonstrated sustained improvement in the signs and symptoms of psoriatic arthritis (PsA) over 2?years in the FUTURE 2 study (NCT01752634). This post hoc analysis assessed the ability of secukinumab to achieve Psoriatic Arthritis Disease Activity Score (PASDAS)-based remission or low disease activity (LDA) through 2?years among patients with PsA in the FUTURE 2 study.

Methods

PASDAS (cut-off scores: remission ≤?1.9; LDA >?1.9 and?<?3.2; Moderate Disease Activity ≥?3.2 and?<?5.4; and high disease activity [HDA]?≥?5.4) was assessed in the overall population (tumour necrosis factor inhibitor [TNFi]-naïve and TNFi-experienced), in patients stratified by prior TNFi use and by disease duration at weeks 16, 52 and 104. The impact of secukinumab on individual PASDAS core components and on the relationship between PASDAS states and patient-reported outcomes (PROs), including physical function, health-related quality of life (HRQoL) and work productivity, were also assessed. Data for the approved doses of secukinumab (300 and 150?mg) are reported. PASDAS scores and core components were reported as observed, and PROs were analysed using mixed models for repeated measures.

Results

In the overall population, PASDAS remission and LDA were achieved in 15.6% and 22.9%, respectively, of patients treated with secukinumab 300?mg and in 15.2% and 19.2%, respectively, in the secukinumab 150?mg group versus 2.3% and 13.8%, respectively, with placebo at week 16. In the TNFi-naïve group, a higher proportion of patients achieved remission?+?LDA at week 16 with secukinumab 300 and 150?mg (46.2% and 42.9%, respectively) versus placebo (17.5%), with corresponding responses in TNFi-experienced patients being 22.6% and 19.4% versus 13.3%. Remission/LDA responses with secukinumab were sustained through 2?years. Patients achieving remission/LDA reported greater improvements in PROs than patients in HDA through 2?years.

Conclusions

Secukinumab-treated patients achieved higher PASDAS-defined remissions or LDA compared with placebo at week 16, which were sustained through 2?years. Remission/LDA was achieved by both TNFi-naïve and TNFi-experienced patients treated with secukinumab, with higher rates in TNFi-naïve patients. Secukinumab-treated patients achieving remission/LDA reported significantly greater improvements in PROs, including physical function and different dimensions of health-related quality of life and work, than patients in HDA.

Trial registration

ClinicalTrials.gov, NCT01752634. Registered on December 19, 2012.EUDRACT, 2012-004439-22. Registered on December 12, 2012.

     

Bayesian adaptive survey protocols for resource management

Transparency in resource management decisions requires a proper accounting of uncertainty at multiple stages of the decision-making process. As information becomes available, periodic review and updating of resource management protocols reduces uncertainty and improves management decisions. One of the most basic steps to mitigating anthropogenic effects on populations is determining if a population of a species occurs in an area that will be affected by human activity. Species are rarely detected with certainty, however, and falsely declaring a species absent can cause improper conservation decisions or even extirpation of populations. We propose a method to design survey protocols for imperfectly detected species that accounts for multiple sources of uncertainty in the detection process, is capable of quantitatively incorporating expert opinion into the decision-making process, allows periodic updates to the protocol, and permits resource managers to weigh the severity of consequences if the species is falsely declared absent. We developed our method using the giant gartersnake (Thamnophis gigas), a threatened species precinctive to the Central Valley of California, as a case study. Survey date was negatively related to the probability of detecting the giant gartersnake, and water temperature was positively related to the probability of detecting the giant gartersnake at a sampled location. Reporting sampling effort, timing and duration of surveys, and water temperatures would allow resource managers to evaluate the probability that the giant gartersnake occurs at sampled sites where it is not detected. This information would also allow periodic updates and quantitative evaluation of changes to the giant gartersnake survey protocol. Because it naturally allows multiple sources of information and is predicated upon the idea of updating information, Bayesian analysis is well-suited to solving the problem of developing efficient sampling protocols for species of conservation concern. © 2011 The Wildlife Society.     

Scanning electron microscopy of cloned astrocytic lines derived from ethylnitrosourea-induced rat gliomas

Six cloned astrocytoma cell lines derived from four ethylnitrosourea-induced F-344 rat gliomas were viewed by scanning electron microscopy in vitro, and two were examined in vivo after transplantation to the intracerebral site. All clones consisted of stellate cells that were reasonably homogeneous within individual glioma lines. Cell membrane features common to all tumor lines included microvilli, blebs, ruffles, and miniridges, mainly confined to perikarya, and filopodia emanating chiefly from cell processes. One cell line demonstrated a profuse, and another cell line a moderate, degree of microvillous development and cell surface roughening, which in one tumor correlated with rapid in vitro cell doubling time. Both cell lines maintained these topographical appearances when transplanted into brain. These results extend the SEM observations of astrocytomas, particularly in cloned ethylnitrosourea-induced tumors in rats. The confirm that distinct variations in cell membrane topography do occur among tumors of this type, probably irrespective of their origin in humans or rats, and irrespective of their mode of genesis as spontaneous, chemically-induced, or virally-induced tumors.     

Characterization of an anaerobic marine microbial community exposed to combined fluxes of perchlorate and salinity

Applied Microbiology and Biotechnology - The recent recognition of the environmental prevalence of perchlorate and its discovery on Mars, Earth’s moon, and in meteorites, in addition to its...     

Immune protection against experimental autoimmune encephalomyelitis: optimal conditions and analysis of mechanism

Protection against experimental autoimmune encephalomyelitis (EAE) was studied in the guinea pig and the Lewis rat. Basic protein of myelin (BPM) injected in incomplete Freund's adjuvant (IFA) gave solid protection against subsequent challenge with normally encephalitogenic doses of BPM in complete Freund's adjuvant (CFA). Protection depended on the amount of BPM in IFA injected and on the duration of the interval between protection and encephalitogenic challenge with BPM in CFA. Notably, protection was long lasting; it remained demonstrable, to some degree for 52 weeks in guinea pig and 32 weeks in rats, these being the longest intervals tested.Protection could not be correlated with serum antibody levels to BPM, and was afforded in the guinea pig by the injection, in IFA, of a synthetic peptide matching residues 112–122 of human BPM; this peptide produced no detectable serum antibody to BPM. Protected guinea pigs had intact cell-mediated immunity to BPM, as measured by inhibition of macrophage migration in vitro. The mechanism of protection may involve the production, following injection of BPM in IFA, of a class of suppressor thymic lymphocytes capable of overriding otherwise encephalitogenic thymic lymphocytes.     

Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction

The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.