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Effects of adenine nucleosides and nucleotides on neuromuscular transmission to the prostatic stroma of the rat.

1. The aim of this study was to investigate the effects of adenine nucleosides and nucleotides on contractility of the smooth muscle of rat prostate gland. 2. Nerve terminals within rat isolated prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine had no effect on baseline smooth muscle tone but concentration-dependently inhibited electrically-evoked contractile responses. The relative order of potency was ATP congruent with AMP congruent with adenosine>ADP. 3. The inhibition by ATP and adenosine of field stimulation-induced contractions in the rat prostate was antagonized by 8-phenyltheophylline (10 microM), but not by suramin (100 microM) and only slightly by reactive blue 2 (5 microM). 4. The adenosine metabolizing enzyme adenosine deaminase (0.1 unit ml(-1)) inhibited the inhibitory effects of ATP and adenosine. The P2 purinoceptor agonist 2-methylthio ATP (10 nM - 0.1 mM), had no effect on field stimulation-induced contractions of the rat prostate. 5. ATP and adenosine did not modify the contractile responses of the rat prostate to exogenously added noradrenaline (10 microM). 6. Inhibitory concentration-response curves to a number of adenosine analogues with differing stabilities and selectivities for the different adenosine receptors yielded a relative rank order of agonist potency of: N(6)-cyclopentyladenosine (CPA)>N(6)-cyclohexyladenosine (CHA) congruent with (-)-N(6)-(2-phenylisopropyl)-adenosine (R-PIA) congruent with 5'-(N-ethylcarboxamido)-adenosine (NECA)>(+)-N(6)-(2-phenylisopropyl)-adenosine (S-PIA)>2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-ade nosine (CGS 21680). 7. These results indicate that adenine nucleoside and nucleotide induced inhibition of electrically-evoked contractions in the rat prostate occurs through activation of adenosine but not ATP receptors. The relative order of potency of adenosine analogues is consistent with activation of receptors of the A(1)-adenosine receptor subtype. These receptors appear to be prejunctional.

Adenosine Diphosphate↗

Template synthesis of tungsten complexes with saturated N-heterocyclic carbene ligands.

Tungsten complex with a coordinated 2-azidoethyl isocyanide ligand reacts with PMe3 at the azido function to give a complex with a coordinated iminophosphorane which upon hydrolysis of the P=N bond yields a complex with an NH,NH-stabilized N-heterocyclic carbene ligand, 7; alkylation of the carbene ring nitrogen atoms gives a complex with an N,N'-dialkylated imidazolidin-2-ylidene ligand, 8 .

Journal Article↗

Characterization and modulation of the hierarchical self-assembly of nanostructured DNA tiles into supramolecular polymers.

We present a set of DNA supramolecular architectures based on the polymerization of discrete DNA tiles having the shape of parallelograms and designed to have a one-dimensional inter-tile connectivity. Tiles bind to each other with two connections, which have different thermal stabilities. We discuss how this difference in stability implies that the same monomeric tile can yield supramolecular polymers of different shapes just by changing the polymerization conditions. We show how this system reacts to external stimuli by interconverting between some of its possible states. Concurrently, we show how performing the polymerization on a surface can influence its outcome.

Base Sequence↗

Analyzing intramolecular dynamics by fast Lyapunov indicators.

We report an analysis of intramolecular dynamics of the highly excited planar carbonyl sulfide below and at the dissociation threshold by the fast Lyapunov indicator method. By mapping out the variety of dynamical regimes in the phase space of this molecule, we obtain the degree of regularity of the system versus its energy. We combine this stability analysis with a periodic orbit search, which yields a family of elliptic periodic orbits in the regular part of phase space and a family of in-phase collinear hyperbolic orbits associated with the chaotic regime.

Journal Article↗

Internal packing conditions and fluctuations of amino acid residues in globular proteins.

In order to investigate the environmental conditions of amino acid residues in protein molecules, four kinds of packing studies (atomic, geometric, hydrophobic and hydration) were formulated and tested on two proteins; bovine pancreatic trypsin inhibitor (BPTI) and bovine pancreatic ribonuclease S (RNase S). The inter-relationship of these packings on the fluctuations of amino acid residues was analysed by comparing the packing results with the dynamical studies, such as the root-mean-square-deviation values of atomic displacements obtained from the trajectories of molecular dynamics simulation, temperature factor information from crystal structures and residue fluctuations in proteins from continuum model. These analyses yield information about the most fluctuating and most stabilizing residue sites. Comparison of the results obtained by these methods indicate a good agreement, specifying an inverse correlation between the residue packing and fluctuations. This kind of study is helpful in identifying the specific residue sites such as nucleation, receptor binding and antigenic determining sites which in a way indirectly correlates with the functional residues in protein molecules.

Amino Acids↗

A novel protecting strategy for internucleosidic phosphate and phosphorothioate groups.

The utility of 2-(N-isopropyl-N-anisoylamino)ethyl group for protection of internucleosidic phosphate linkages in oligonucleotide synthesis was studied. The group demonstrated high coupling yields, favorable deprotection kinetics and a high hydrolytic stability of phosphoramidite building blocks. The mechanism of deprotection was established using a model phosphate triester.

Oligonucleotides↗

Butadiene monoxide and deoxyguanosine alkylation products at the N7-position.

3,4-Epoxy-1-butene, an active metabolite of 1,3-butadiene, was reacted with guanosine, deoxyguanosine and calf thymus DNA. The products were isolated and positively identified using various spectroscopic techniques. Treatment of calf thymus-DNA with 3,4-epoxy-1-butene yielded two N7-guanine adducts of equal stability. Depurination by neutral hydrolysis showed that 7-(2-hydroxy-3-buten-1-yl)guanine (compound I) was formed in greater quantities compared to its regioisomer 7-(1-hydroxy-3-buten-2-yl)guanine (compound II); spontaneous depurination experiments showed that compound I was released in the highest proportion. The circular dichroism spectral studies with R and S 3,4-epoxy-1-butene revealed that the reaction mechanism at aqueous neutral pH media is more similar to SN2-type rather than SN1-type. The HPLC-electrochemical detection method used to carry out the DNA alkylation study provides a rapid and sensitive quantitation of N7 guanine adducts in biological fluids. This serves as a useful tool for further human biomonitoring experiments.

Alkylation↗

Improved folding of apo-retinol-binding protein in the periplasm of Escherichia coli: positive influences of dsbC coexpression and of an amino acid exchange in the vitamin A binding site.

The in vivo folding of the serum retinol-binding protein (RBP), a representative of the lipocalin structural family, is known to be complex. In order to gain insight into the essential steps along its folding pathway the heterologous production of the functional protein in Escherichia coli was investigated. Simultaneous overexpression of the bacterial dsbC gene, which codes for a periplasmic thiol-disulphide oxidoreductase, prevented the formation of soluble RBP variants with non-native disulphide bonds that were otherwise observed. Although the coexpression of dsbC had furthermore a stabilizing effect on the cell viability, the relative yield of the solubly produced RBP was not much better. In an attempt to enhance its folding efficiency, a favourable point mutation in the inner part of the retinol-binding pocket was predicted. Replacement of the polar Gln117 with an lie side chain seemed not only to relieve the unfavourable energetics of the carboxamide group in the environment of predominantly non-polar residues but also to fill an adjacent cavity in the hydrophobic core. Indeed, this single substitution reproducibly resulted in a more than threefold increase in the amount of functional recombinant RBP. Ligand binding experiments showed that the affinity of this mutant for retinol was slightly enhanced. Kinetic measurements revealed that this was due to a higher association rate whereas the dissociation of the complex with retinol was essentially unaffected. Although the question remained why nature did not select this obviously beneficial mutation, our results demonstrate that the folding pathway of a lipocalin can be optimized by protein engineering.

Amino Acid Substitution↗

Closed Ender nailing of femur fractures in older children.

Twenty-eight patients ranging from 8 to 13 years old were treated at Childrens Hospital of Alabama for diaphyseal femur fractures. We studied two groups: 14 patients (Group I) were treated by conventional 90-90 balanced skeletal traction with late spica cast application; 14 patients (Group II) with multiple associated injuries or hyperkinetic problems were treated with closed retrograde Ender nailing. Groups I and II compared closely with regard to age, sex, fracture type and location, and mechanism of injury. The alignment, leg length, length of hospitalization, and length of therapy were analyzed. Our experience indicates that closed retrograde Ender nailing of diaphyseal femur fractures is an ideal technique where operative reduction and stabilization are indicated in older children. The technique yields results superior to conventional methods of treatment, with the advantage of early discharge from the hospital and return to school.

Bone Nails↗

Reoperation in patients after anterior cervical plate stabilization in degenerative disease.

STUDY DESIGN: Consecutive case retrospective chart review. OBJECTIVES: First, to assess whether the number of' patients requiring a second cervical surgical intervention was changed as a result of using anterior cervical plate stabilization, and second, to determine the additional risks and/or benefits associated with the hardware implantation. SUMMARY OF BACKGROUND DATA: The optimal technique of performing stabilization, arthrodesis, and alignment of a cervical segment after discectomy with neural decompression in degenerative disease has yet to be determined. METHODS: The charts of 402 patients who had undergone an anterior cervical discectomy and arthrodesis for degenerative disease performed both with and without anterior cervical plate stabilization were reviewed, and reoperation data were compiled. The average follow-up time was 3.8 years (range, 1.5-9.4 years). RESULTS: Of 365 patients with 1- or 2-level cervical arthrodesis, 22 required a second surgical intervention (20 bone alone, 2 with anterior cervical plate stabilization). The Log-Rank test, which uses all patients and their total follow-up periods, was statistically significant favoring anterior cervical plate stabilization at one and two levels (P = 0.015). CONCLUSIONS: The addition of anterior cervical plate stabilization in one- and two-level cervical degenerative disease supplements the internal stabilization initially provided by the bone graft, and yields a lower reoperation rate.

Adult↗

New magnetic order in buried native iron oxide layers.

The properties of a magnetically ordered buried Fe oxide layer are presented. This oxide has a room-temperature magnetization exceeding that of Fe3O4 by 42% and of gamma-Fe2O3 by 89%. The oxide consists of a component (70%) with a net moment of 2.0 micro(B)/Fe ion, while the remaining spins yield no net moment. The oxide magnetization is stabilized in part by the proximate Fe metal.

Journal Article↗

Alasan, a new bioemulsifier from Acinetobacter radioresistens.

Acinetobacter radioresistens KA53, isolated by enrichment culture, was found to produce an extracellular, nondialyzable emulsifying agent (referred to as alasan) when grown on ethanol medium in a batch-fed reactor. The crude emulsifier was concentrated from the cell-free culture fluid by ammonium sulfate precipitation to yield 2.2 g of emulsifier per liter. Alasan stabilized a variety of oil-in-water emulsions, including n-alkanes with chain lengths of 10 or higher, alkyl aromatics, liquid paraffin, soybean and coconut oils, and crude oil. Alasan was 2.5 to 3.0 times more active after being heated at 100 degrees C under neutral or alkaline conditions. Emulsifying activity was observed over the entire pH range studied (pH 3.3 to 9.2), with a clear maximum at pH 5.0. Magnesium ions stimulated the activity both below (pH 3.3 to 4.5) and above (pH 5.5 to 9.3) the pH optimum. Alasan activity was higher in 20 mM citrate than in 20 mM acetate or Tris-HCl buffer. Preliminary chemical characterization of alasan indicated that it is a complex of an anionic, high-molecular-weight, alanine-containing heteropolysaccharide and protein.

Acinetobacter↗

Bacteremia after genitourinary tract manipulation: bacteriological aspects and evaluation of various blood culture systems.

A total of 300 patients undergoing various types of urological procedures was studied for incidence of bacteremia. An osmotically stabilized anaerobic broth with sodium polyanethol sulfonate (Liquoid) yielded more positive blood cultures than any other culture system and was also the best system by far for recovery of anaerobes. The membrane filter showed faster growth and, therefore, facilitated faster identification of the infecting organism. There was a 31% incidence of bacteremia in the patients having transurethral resection of the prostate, 17% in the cystoscopy group, 24% in the urethral dilation group, and 8% in the urethral catheterization group. The organisms found most frequently isolated in blood cultures were enterococci and Klebsiella pneumoniae. Notable were a relatively large number of anaerobes and two protoplasts. The major source of the bacteremia was previous urinary tract infection, but evidence is presented which indicates that the prostate gland and the normal urethral flora are other significant sources.

Anaerobiosis↗

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.

Acinetobacter↗

Control of VP16 translation by the herpes simplex virus type 1 immediate-early protein ICP27.

Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and beta-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.

Actins↗

The fetal/adult acetylcholine receptor antibody ratio in mothers with myasthenia gravis as a marker for transfer of the disease to the newborn.

High anti-fetal/anti-adult muscle anti-acetylcholine receptor (AChR) antibody (Ab) titer ratio is predictive of the occurrence of neonatal myasthenia gravis in a first child. The aim of the present study was to determine whether the ratio between the levels of antibodies is an intrinsic property of the mother's sera or varies with physiologic status such as pregnancy. We performed a longitudinal study of the levels of Ab directed against both fetal and adult AChRs and the ratio between them in 11 mothers with myasthenia gravis (MG). Sera were taken during, before, and after pregnancy. Absolute levels of Ab varied considerably during the time of observation as indicated by analyzing the maximum change between any two sample times during the study (adult mean percentage change 45.9 +/- 26.4; fetal 42.51 +/- 22.05). In contrast to this, the anti-fetal/anti-adult muscle AChR Ab titer ratio was much less variable (mean percentage change 16.66 +/- 10.11; p < 0.0033). The levels of the two Ab types yielded a correlation of 0.918, consistent with the stability of the ratio between them. This stability of ratio has practical value in the management of pregnancy and infant care in mothers with MG because the ratio at any time, before or during pregnancy, will predict whether the child will contract neonatal MG. We determined this for the first child, but further studies are necessary to establish if this remains true for subsequent pregnancies.

Antibodies↗

Synthesis of new synthons for organofluorine compounds from halothane containing sulfur functional groups.

To develop new synthons for the syntheses of organofluorine compounds, the treatment of Halothane, 2-bromo-2-chloro-1,1,1-trifluoroethane, (1) with 4-methylbenzenethiol (2) in the presence of sodium hydride gave 1-chloro-2,2,2-trifluoroethyl 4-methylphenyl sulfide (3), which was oxidized with m-chloroperbenzoic acid (m-CPBA) to the corresponding sulfoxide (4) and sulfone (5). Reaction of 3 and 5 with allyltributyltin in the presence of 2,2'-azobis(isobutyronitrile) (AIBN) gave 1-(trifluoromethyl)-3-butenyl compounds (9, 11). Sulfoxide 4 was decomposed in this condition. The treatment of 3 with allyltrimethylsilane in the presence of Lewis acids gave 1-(trifluoromethyl)-3-butenyl compounds (9) in good yield. This result suggests that 4-methylphenylthio substituent stabilizes the alpha-carbocation effectively, though the trifluoromethyl group destabilizes it strongly. Aromatic compounds similarly reacted with 3 in the presence of titanium(IV) chloride to give 2-aryl-1,1,1-trifluoro-2-(4-methylphenylthio)ethanes. Thus, sulfur compounds derived from Halothane were found to be useful new synthons for organofluorine compounds.

Halothane↗

Optimizing splinted ligation of highly structured small RNAs.

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73 mer tRNA, the other a 49 mer synthetic ribozyme. A stable LCC could be isolated and turned over with>95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

Base Sequence↗