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At least 703 records · Page 39Linked to original sources
PubMed1996

Influence of technetium-99m-labeling conditions on physico-chemical and related biological properties of an acylated poly-galactosidic macrophage targeting agent for inflammation imaging.

The potential of 99m-Tc-J001 for the investigation of inflammatory lesions via the targeting of recruited macrophages (M phi) has already been documented in several experimental models and in human diseases. To achieve a functional imaging of inflammation via M phi targeting, minimal labeled colloid content and high in vivo stability of 99mTc-J001 are essential. The actual specificity of such scintigraphy is closely dependent upon the radiolabeling of only the J001 molecules available for M phi targeting. To develop an appropriate radiopharmaceutical kit, optimization of the labeling conditions was achieved from a series of pilot formulations that were evaluated for radiolabeling efficiency and both in vitro and in vivo 99mTc-J001 stability. Colloids were characterized using autocorrelation spectroscopy and multiangle laser-light scattering, radioactive colloid content of the formulations being deduced from biodistribution studies. This work has made possible the definition of a formulation exhibiting a radiolabeling yield > 97.0%, associated with in vivo stability and minimal colloid formation, thus greatly enhancing the specificity of such macrophage scintigraphy.

Animals↗
PubMed1992

Identification of Clostridium histolyticum collagenase hyperreactive sites in type I, II, and III collagens: lack of correlation with local triple helical stability.

The class I and II Clostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the alpha 1(I), alpha 2(I), alpha 1(II), and alpha 1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.

Amino Acid Sequence↗
PubMed1992

Efficiency of the 5'-terminal sequence (omega) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli.

Recent studies have demonstrated that the 5' leader (omega sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes. In order to estimate the efficiency of omega to initiate translation of mRNA in Escherichia coli, in comparison to the Shine-Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E. coli expression vectors containing one constitutive promoter (P1) but different translation-initiation sites (S/D or omega delta 3 sequence, respectively). The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA. We report that substitution of omega delta 3 for S/D decreases the yield of expressed protein 4-1900-fold and the content of gene-specific mRNA is decreased by about sevenfold. However, in comparison with the S/D sequence, the level of protein expressed under the translational control of omega delta 3 is less sensitive to changes in the 5' coding region. We also report that the omega sequence contains a region of 10-12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E. coli, Eikenella corrodens and Xenopus laevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.

Amino Acid Sequence↗
PubMed1996

Genetic (co)variances among birth weight, 200-day weight, and postweaning gain in composites and parental breeds of beef cattle.

Genetic and environmental (co)variances for birth weight, adjusted 200-d weight, and postweaning gain were estimated in nine parental and three composite populations of beef cattle. The parental breeds were Angus (A), Braunvieh (B), Charolais (C), Gelbvieh (G), Hereford (H), Limousin (L), Pinzgauer (P), Red Poll (R), and Simmental (S). The composites were MARC I (1/4 B, 1/4 C, 1/4 L, 1/8 H, 1/8 A), MARC II (1/4 G, 1/4 S, 1/4 H, 1/4 A), and MARC III (1/4 R, 1/4 P, 1/4 H, 1/4 A). Heritabilities of additive direct genetic effects for birth weight (.50) and postweaning gain (.49) were greater than for 200-d weight (.32). Heritabilities of additive maternal effects of .09 for birth weight and .10 for 200-d weight were much smaller than direct effect heritabilities. Heritabilities were larger in composites than in parental breeds for additive direct effects of all three traits but smaller for maternal 200-d weight. Correlations were high and positive for direct genetic effects of the three weight traits and higher in composites than in the parental breeds. Correlations between direct and maternal genetic effects for both birth weight and 200-d weight were near zero. Some differences in variances among populations were correlated with differences in weight and milk yield. Heavier populations had larger variances, supporting the use of logarithmic transformation of weights to stabilize variances among genetic groups. Increased average milk yield was correlated with decreased phenotypic variance of 200-d weight. Average milk yield was also implicated in the expression of direct and maternal genetic effects for 200-d weight and their covariance. Comparison of univariate and multivariate estimates of genetic variances suggested that it is important to include birth weight in multivariate analyses of all weight traits to account for increased preweaning mortality of calves with extremely heavy or light birth weights. Based on heritability estimates, within-herd selection in composites should be at least as effective as in purebreds. Some differences among populations in genetic parameters were indicated, especially maternal 200-d weight and its correlations with other traits.

Animals↗
PubMed2004

The folding state of the lumenal loop determines the thermal stability of light-harvesting chlorophyll a/b protein.

The major light-harvesting protein of photosystem II (LHCIIb) is the most abundant chlorophyll-binding protein in the thylakoid membrane. It contains three membrane-spanning alpha helices; the first and third one closely interact with each other to form a super helix, and all three helices bind most of the pigment cofactors. The protein loop domains connecting the alpha helices also play an important role in stabilizing the LHCIIb structure. Single amino acid exchanges in either loop were found to be sufficient to significantly destabilize the complex assembled in vitro [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093. Mick, V., Eggert, K., Heinemann, B., Geister, S., and Paulsen, H (2004) Biochemistry 43, 5467-5473]. This work presents an analysis of such point mutations in the lumenal loop with regard to the extent and nature of their effect on LHCIIb stability to obtain detailed information on the contribution of this loop to stabilizing the complex. Most of the mutant proteins yielded pigment-protein complexes if their reconstitution and/or isolation was performed under mild conditions; however, the yields were significantly different. Several mutations in the vicinity of W97 in the N-proximal section of the loop gave low reconstitution yields even under very mild conditions. This confirms our earlier notion that W97 may be of particular relevance in stabilizing LHCIIb. The same amino acid exchanges accelerated thermal complex dissociation in the absence of lithium dodecyl sulfate (LDS) and raised the accessibility of the lumenal loop to protease; both effects were well correlated with the reduction in reconstitution yields. We conclude that a detachment of the lumenal loop is a possible first step in the dissociation of LHCIIb. Dramatically reduced complex yields in the presence but not in the absence of LDS were observed for some but not all mutants, particularly those near the C-proximal end of the loop. We conclude that complex stabilities in the absence and in the presence of LDS do not correlate and most likely are determined by different structural characteristics, at least in LHCIIb but maybe also in other membrane proteins.

Amino Acid Substitution↗
PubMed1974

Rapid loss of translatable messenger RNA of phosphoenolpyruvate carboxykinase during glucose repression in liver.

The rate of synthesis of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) in rat liver decreased with a half-life of 30 min after fasted rats were refed either a chow diet or glucose. A requirement for both glucose and insulin to bring about this rapid deinduction was shown, as well as the ability of dibutyryl adenosine 3':5'-cyclic monophosphate to block the decrease in enzyme synthesis. Estimates of the stability of messenger RNA of phosphoenolpyruvate carboxykinase were made by using the inhibitors actinomycin D and cordycepin to block further messenger RNA synthesis, and then measuring the decrease in specific enzyme synthesis. It is suggested that the use of actinomycin D yields an overestimation of the template stability. The results with cordycepin imply that the enzyme messenger RNA has a short half-life of approximately 1 hr. Thus, it is possible that deinduction may proceed by way of a decrease in messenger RNA production, leading to a rapid fall in the synthesis of phosphoenolpyruvate carboxykinase.

Animals↗
PubMed1996

Calcium-dependent oligonucleotide antagonists specific for L-selectin.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

Base Sequence↗
PubMed1999

Synthesis of [99mTc]ethylenedicysteine-colchicine for evaluation of antiangiogenic effect.

Angiogenesis is in part responsible for tumor growth and the development of metastasis. Radiolabeled angiongenesis inhibitors would be useful to assess tumor microvasculature density. Colchicine (COL), a potent antiangiogenic agent, is known to inhibit microtubule polymerization and cell arrest at metaphase. This study aimed to develop 99mTc-labeled COL (EC-COL) using ethylenedicysteine (EC) as a chelator to assess tumor microvascular density. EC was conjugated to trimethylcolchicinic acid using N-hydroxysuccinimide and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling agents with a yield of 50-60%. In vivo stability was analyzed in rabbit serum at 0.5-4 h. Tissue distribution and planar imaging studies of [99mTc]EC-COL were evaluated in breast tumor-bearing rats at 0.5, 2 and 4 h. The data was compared to that using [99mTc]EC (control). The radiochemical yield of [99mTc]EC-COL was greater than 95%. [99mTc]EC-COL was stable in rabbit serum. In vivo biodistribution of [99mTc]EC-COL in breast tumor-bearing rats showed increased tumor-to-blood (0.52+/-0.12 to 0.72+/-0.07) and tumor-to-muscle (3.47+/-0.40 to 7.97+/-0.93) ratios as a function of time. Conversely, tumor-to-blood values showed a time-dependent decrease with [99mTc]EC over the same time period. Planar images confirmed that the tumors could be visualized clearly with [99mTc]EC-COL from 0.5 to 4 h. [99mTc]EC-COL may be useful to assess antiangiogenic and therapeutic effects during chemotherapy.

Angiogenesis Inhibitors↗
PubMed1998

Slowing of peroxynitrite decomposition in the presence of mannitol and ethanol.

The kinetics of peroxynitrite decomposition in the presence of the hydroxyl radical scavengers mannitol and ethanol were studied by stopped-flow spectrophotometry. Mannitol and ethanol decreased the rate of peroxynitrite decomposition in a concentration-dependent manner, following a hyperbolic function. The decreases in peroxynitrite decay rates were observed all throughout the pH range 5.8 to 8.0. In the presence of 100 mM mannitol or ethanol, the first-order rate constant for peroxynitrite decomposition changed from 1.25 +/- 0.01 s-1 at 25 degrees C, to values of 0.83 +/- 0.01 s-1 and 0.95 +/- 0.01 s-1, respectively. One explanation for this decrease in the rate of peroxynitrite decay with mannitol and ethanol could be a stabilizing effect of the substrate by hydrogen bonding with peroxynitrite, analogous to what has been recently proposed for hydrogen peroxide (Alvarez, B., Denicola, A. and Radi, R. Chem. Res. Toxicol. 8:859-869; 1995). In this sense, kinetic data fitted a mechanism implying fast equilibria between peroxynitrite anion and peroxynitrous acid with the substrates to form the corresponding complexes. The equilibrium constants of complex dissociation were estimated to be (6.7 +/- 0.9) x 10(-3) M and (9.6 +/- 1.5) x 10(-3) M for mannitol and ethanol, respectively. When bonded to mannitol or ethanol, peroxynitrous acid could ionize, too, or decompose at a slower rate than in the absence of substrate, in part to a reactive intermediate which performs oxidations. While mannitol and ethanol inhibit oxidation and nitration processes that occur through the reaction of secondary reactive intermediates of peroxynitrite with target molecules, up to 0.5 M mannitol or ethanol failed to inhibit cysteine oxidation by peroxynitrite at pH 7.4 and 25 degrees C. Thus, the formation of stabilizing complexes would not divert the reaction yield of direct, second order reactions such as thiol oxidation, but highlights the importance of hydrogen bonding and solvent effects on peroxynitrite stability.

Buffers↗
PubMed2002

An improved synthesis of cyclopropanes from stabilized phosphonates and 1,2-dioxines.

Addition of stabilized Horner-Wadsworth-Emmons (HWE) phosphonates to substituted 1,2-dioxines leads to diastereomerically pure di- and trisubstituted cyclopropanes in high yields and represents a viable alternative to ylides in the cyclopropanation reaction involving 1,2-dioxines. While yields are comparable, reaction times with these stabilized phosphonates were accelerated and the diastereoselectivity for this cyclopropanation reaction was significantly greater than for the previously reported examples employing ylides.

Journal Article↗
PubMed1995

The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation.

We have used either Escherichia coli or T7 RNA polymerase to transcribe in E. coli a series of lacZ genes that differ in the nature of their ribosome binding sites (RBS). Each T7 RNA polymerase transcript yields from 15- to 450-fold less beta-galactosidase than its E. coli polymerase counterpart, the ratio being larger when weaker RBS are used. The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E. coli RNA polymerase transcripts. T7 RNA polymerase transcribes the lacZ gene approximately 8-fold faster than the E. coli enzyme. We propose that this higher speed unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E. coli enzyme is used. This leads to degradation of the T7 transcript, unless the leading ribosome comes in time to shield the cleavage site: the weaker the RBS, the lower this probability and the more severe the inability of T7 RNA polymerase transcripts for beta-galactosidase synthesis.

Base Sequence↗
PubMed2001

UV laser photolysis of DNA: effect of duplex stability on charge-transfer efficiency.

The distribution of the final base damage was determined within isolated DNA exposed to pulses of 266 nm laser light. Studied lesions included oxidation products arising from biphotonic ionization of DNA bases and pyrimidine dimeric photoproducts arising from monophotonic processes. The distribution of the latter class of damage was found to be correlated with the stability of the DNA duplex. The quantum yield for formation of 8-oxo-7,8-dihydroguanine was much higher than that of other oxidized nucleosides arising from the degradation of thymine and adenine. This observation, together with the shape of the intensity dependence curves, provided evidence for the occurrence of charge-transfer processes within DNA. In addition, increase in the ionic strength of the irradiated DNA and stabilization of the DNA duplex were found to induce a drastic decrease in the yield of thymine and adenine oxidation products. Concurrently, an increase in the yield of 8-oxo-7,8-dihydroguanine was observed. This was rationalized in terms of an increase in the overall charge-transfer efficiency. Therefore, it may be concluded that stabilization of the double-helix favors charge-transfer process toward guanine bases.

Animals↗
PubMed1982

[On the stability of phenylmercury salts. Part 50: Contributions to problems concerning the use of plastic receptacles for liquid pharmaceuticals (author's transl)].

The authors describe a dithizone method for the determination of phenylmercury salts that yields easily reproducible results on examining the stability of these preserving agents. Aqueous solutions of phenylmercury salts are instable already in glass bottles, but they are instable to a much greater extent in low-density polyethylene bottles. Their degradation is accelerated in the presence of chloride ions. When stored under nitrogen, the phenylmercury salt solutions show lower losses of preserving agents than when stored under normal air. The ultra-violet extinction of phenylmercury salt solutions increases during storage in glass bottles, and decreases during storage in plastic bottles. In plastic bottles, the degradation is associated with sorption.

Chemical Phenomena↗
PubMed1989

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.

Cell Line↗
PubMed1985

Polysaccharides and food processing.

The rôle of polysaccharides during processing and for the quality of foods is discussed. Starch is the most important energy source for man. Most other polysaccharides are not metabolized for energy, but play an important rôle as dietary fibres. Pectins, alginates, carrageenans, and galactomannans are discussed as functional food additives in relation to their structure and their rheological behaviour, stability and interactions. Endogenous polysaccharides of fruits and vegetables and in products derived from them are responsible for such phenomena as texture (changes), press yields, ease of filtration and clarification, cloud stability, and mouth feel. To achieve desirable properties, the action of endogenous enzymes on polysaccharides must be inactivated and/or exogenous enzymes added as processing aids. This is also true for overcoming haze phenomena in clear juices or to break down undesirable microbial polysaccharides. Dough properties for bread baking can be improved by enzymic breakdown of a restrictive pentoglycan network. Network formation may come about by oxidative coupling of phenol rings of ferulic acid bound to hemicelluloses by ester links. Gels may be made by inducing oxidative coupling in natural or synthetic systems. Stagnation in development of new polysaccharide food additives is ascribed to difficulties in obtaining government approval for food use.

Carbohydrate Conformation↗
PubMed2006

Determining the ratio of the Gaussian curvature and bending elastic moduli of phospholipids from Q(II) phase unit cell dimensions.

A method is presented for measuring M, the ratio of the Gaussian (saddle splay) elastic modulus to the bending elastic modulus of a lipid monolayer. The ratio M is determined from measurements of the equilibrium bicontinuous inverted cubic (Q(II)) phase unit cell size in excess water as a function of temperature. The analysis includes the effect of a curvature elastic term that is second-order in the Gaussian curvature, K. Preliminary results using data on DOPE-Me validate the method. The fitted value of M is within 8% of the value estimated in an earlier treatment. The method can be used to measure changes in M due to addition of exogenous lipids and peptides to a host lipid system. The Gaussian elastic modulus has a substantial effect on the stability of fusion intermediates (stalks, hemifusion diaphragms, and fusion pores). Studying the effects of peptides and different lipids on M via this method may yield insights into how fusion protein moieties stabilize intermediates in membrane fusion in vivo. The contribution of the K2 curvature elastic term to the free energy of Q(II) phase and fusion pores explains some features of fusion pore stability and dynamics, and some peculiar observations concerning the mechanism of L(alpha)/Q(II) phase transitions.

Animals↗
PubMed2004

High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.

Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.

3' Untranslated Regions↗
PubMed2000

Two-time window and multiangle photon correlation spectroscopy size and zeta potential analysis--highly sensitive rapid assay for dispersion stability.

Polysaccharide-stabilized iron oxide particles are highly potent contrast agents in magnetic resonance imaging. After intravenous injection, the size of these small or ultrasmall particles strongly influences their distribution in the body. Knowledge about the uniformity of particle size distribution within this particle size range is not accessible by laser diffraction (lower edge of detection range), and photon correlation spectroscopy (PCS) data only yield insufficient information, the so-called polydispersity index. A combination of two-time window and multiangle analysis makes detailed characterization of particle size distribution and particle aggregation feasible, which was shown using five different iron oxide dispersions. Additional particle charge characterization yielded conclusions about the type of stabilization present in the dispersion - electrostatic or steric stabilization. Thus, this thorough particle size and charge analysis is a tool for quick detection of broad particle distributions or aggregates.

Ferric Compounds↗