Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate

Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.     

A model to support the search for consensus with conflicting rankings: Multitrident

A general problem of collective decision‐making can be formulated in terms of the N rankings of a discrete set of alternatives expressing the preferences of the N members of the group. Helping to reach a consensus is quite important for team building and organizational development and several contributions were developed by different disciplines like organizational sciences, human resources management and operational research. However, very few models were proposed to support the process of reaching consensus and such models assume a concept of distance between rankings implying an assumption of equal distance between adjacent alternatives. Unfortunately, this hypothesis is hardly acceptable in most cases. In this paper, a model is proposed to search for a ranking in terms of the weights space avoiding the previous assumption and providing useful insights to help decision‐makers adopting such ranking as a consensus decision.     

A regulatory role for recombinase activating genes, RAG-1 and RAG-2, in T cell development

Effects of etoposide (VP-16) and cytosine arabinoside (Ara-C) on the cell cycle of HL-60 and THP-1 cells were studied by flow cytometry using the bromodeoxyuridine (BrdU)/DNA assay technique to investigate the efficacy of VP-16 for monocytic leukemia cells. VP-16 inhibited the proliferation of THP-1 cells more strongly than that of HL-60 cells at any concentrations used at 24 and 48 hr. VP-16 arrested HL-60 and THP-1 cells in the G2/M phase and reduced them in the G0/G1 and early S phase at higher concentrations. There was no significant difference in the percentage of G2/M phase cells at the same concentration between both cells. However, reduction in the G0/G1 and early S phase cells was more marked in THP-1 than HL-60 cells significantly. On the other hand, Ara-C perturbed the cell cycle of HL-60 cells more than that of THP-1 cells at 24 and 48 hr. These results suggest that the effects of VP-16 on the cell cycle may be more intense in THP-1 than HL-60 cells, and support the efficacy of VP-16 for treating monocytic leukemia in vivo.     

Crosstalk between Nutrition,Insulin, Juvenile Hormone,and Ecdysteroid Signaling in the Classical Insect Model,Rhodnius prolixus

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.     

In vitro displacement by rat serum of adsorbed radiolabeled poloxamer and poloxamine copolymers from model and biodegradable nanospheres

Poloxamer 407 and poloxamine 908 have been used by many research groups to modify the surface of both model latex and biodegradable nanospheres, thereby producing nanospheres that have shown reduced protein adsorption in vitro and extended circulation times in vivo. A potential limitation of such systems is the desorption of the copolymer coating layer. We describe a two-stage process to radiolabel poloxamer 407 and poloxamine 908 that has facilitated an investigation into this potential desorption, in vitro. The first stage of the labeling procedure involved the substitution of the terminal hydroxyl groups in each poly(ethylene oxide) (PEO) chain of poloxamer 407 and poloxamine 908 with an amino group. The aminated copolymers were then radiolabeled with 125Iodine Bolton-Hunter reagent. The efficiency of labeling was calculated to be approximately 20% for the tetramine poloxamine 908 and approximately 33% for the diamine poloxamer 407. Remaining free amino groups were then either acetylated, using acetic anhydride, or left in the free amino form. Covalent linkage of the radiolabel to the copolymer was confirmed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The stability of the link between radiolabel and copolymer to hydrolysis was also confirmed; <4% loss of radiolabel occurred from poloxamine 908 after incubation in phosphate-buffered saline (PBS) at 37 degrees C for 8 days. The radiolabeled copolymers (with the free amino groups acetylated) were then used in experiments that have given the first direct evidence that adsorbed copolymers can be displaced by serum proteins in significant amounts from the surface of model and biodegradable nanospheres. The displacement was highly dependent on copolymer-nanosphere compatibility, with up to 78% of 125I tetramine poloxamine 908 being displaced from poly(lactide-co-glycolide) (PLGA) nanospheres in 24 h, compared with 20% displacement of 125I tetramine poloxamine 908 in 24 h from polystyrene nanospheres. These results have direct implication for the future design of drug delivery systems based on coated nanospheres.     

Determination of some physicochemical parameters of microcystins (cyanobacterial toxins) and trace level analysis in environmental samples using liquid chromatography

The present study measured how the detection thresholds for two very brief tone or noise bursts depend on the delay between them. The thresholds for the tone burst pairs systematically increase for increasing delays up to a few milliseconds and then reach a constant value. The tone burst thresholds thus reflect a very short-term interaction between the brief signals. Strikingly, the time constant of the interaction appears to scale inversely to the frequency of the tone bursts. The thresholds for the noise burst pairs, on the other hand, remain approximately constant down to the shortest measured delay of less than 1 ms. In contrast to the concept of intensity integration, these results are interpreted in terms of a temporal overlap of the auditory filter responses elicited by two brief, successively presented signals. In a second experiment it was shown that the observed short-term interaction between two brief signals is sensitive to their relative phases, corroborating the conjecture that the interaction takes place at a level prior to the mechanical to neural transduction in the inner ear.     

How much nitrogen is fixed by biological symbiosis in tropical dry forests? 2. Herbs

Although biological nitrogen fixation (BNF) is considered the main input of N in mature and regenerating native tropical vegetation, it has seldom been quantified. Biomass and N accumulation and fixation were determined for spontaneously occurring herbaceous species in caatinga areas in four regeneration stages (2, 17, 39 and >50?years after abandonment from agricultural use). BNF was estimated using the ¹⁵N natural-abundance method. The 2-year regeneration area had the highest total herb (6,355?kg?ha^?1) and legume (262?kg?ha^?1) biomass production, N stocks (82?kg?ha^?1) and fixed N (5.0?kg?ha^?1). N₂-fixing legumes (nine species in the sampled area) contributed over 97?% of legume biomass in all areas. Macroptilium gracile added the largest amount of N (3.9?kg?ha^?1 in the 2-year regeneration area) because of its large biomass production (205?kg?ha^?1), although it was not the species with the highest proportion of fixed N (76?%). All of the N₂-fixing species obtained large proportions of their N from symbiosis, most of them more than 50?%.However, the amounts of fixed N per unit area were relatively low (0.22?C5.00?kg?ha^?1) because the biomass of N₂-fixing species was always less than 5?% of the total herb biomass.     

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.