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At least 883 records · Page 49Linked to original sources
PubMed1989

Characterization of chemically tritiated microcystin-LR and its distribution in mice.

Chemically tritiated microcystin-LR (spec. act. 194 mCi/mmol), purified to greater than 95% by C-18 reverse-phase high performance liquid chromatography, exhibited the same retention time and ultraviolet absorption profile as unlabeled toxin. Acid-hydrolyzed [3H]-toxin yielded tritiated glutamate and beta-methylasparate. Stability of the nonexchangeable [3H]-toxin in saline and urine was greater than 93% after 42 days stored at 22 degrees, 4 degrees or -20 degrees C. In blood, the breakdown of toxin was temperature- and time-dependent (63% at 22 degrees C, 28 days). Unlabeled toxin was stable for greater than 42 days stored at either 4 degrees or -20 degrees C in saline. The LD50 (mouse, i.p.) of [3H]-microcystin-LR and unlabeled toxin was the same [75 micrograms/kg (65-90) and 65 micrograms/kg (53-80), respectively]. From 3 to 90 min after i.p. injection of 70 micrograms/kg [3H]-microcystin-LR there was a slow absorption of toxin from the peritoneal cavity and efficient accumulation in liver. The elimination half-life of the plasma concentration curve was 29 min. Tritium distribution in tissue at death or 6 hr post injection was similar for all doses (13-101 micrograms/kg). At 101 micrograms/kg, liver contained 56 +/- 1%, intestine 7 +/- 1%, kidney 0.9 +/- 0.2% and carcass 10 +/- 1% of the injected dose. Heart, spleen, lung and skeletal muscle contained less than 1% of the radiolabel.

Animals↗
PubMed1989

An eyecup slice preparation for intracellular recording in vertebrate retinas.

This report describes a new preparation for intracellular recording from the vertebrate retina, the eyecup slice preparation. It consists of a small (2 X 5 mm) strip cut from the posterior wall of the eye and thereby keeps the sclera, pigment epithelium and neural retina in place. Initial results are presented here for two vertebrates: the turtle, Pseudemys scripta elegans, and the toad, Bufo marinus. With conventional microscopy, the histological layers of the retina can be resolved, as well as individual photoreceptors, to provide landmarks for intracellular recording. When superfused, the eyecup slice remains in good condition for many hours and yields intracellular recordings of good quality and stability. Recordings of the light-evoked responses of cones and horizontal cells show that the slice is large enough to preserve the characteristic spatial interactions mediated by the laterally coursing neural networks of the distal retina. Recordings from rods show that full dark adaptation is achieved. Thus, photochemical dark adaptation as well as other normal cellular interactions between the neural retina and pigment epithelium can be preserved in this preparation, in contrast to isolated retinal slice preparations. The eyecup slice preparation might be particularly useful for work on mammalian retinas.

Adaptation, Physiological↗
PubMed1992

Stably inherited killer activity in industrial yeast strains obtained by electrotransformation.

Killer-sensitive strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were transformed by electroinjection using double-stranded RNA isolated from a superkiller strain. Various recipient strains were used: both thermo-resistant and thermo-sensitive as well as mutants of industrial strains. Conversion of respiratory competent (rho+) into respiratory deficient (rho-) strains (mutants) resulted in a significant increase of the yield of electrotransformants and/or of longterm killer stability. Electrotransformation of rho- mutants of distillery and brewery strains resulted in more than 100 clones, which exhibited weak or strong killer activity over some or all of the experimental period of 10 months.

DNA, Mitochondrial↗
PubMed1993

Yeast systems for the expression of heterologous gene products.

Significant advances have been made over the past year in our understanding of some of the critical parameters affecting high-level production of heterologous proteins in yeast. Recent studies of plasmid stability, promoter strength and secretion efficiency are yielding potential improvements in expression.

Biotechnology↗
PubMed1994

Effects of immunosuppressants FK506 and rapamycin on the heterooligomeric form of the progesterone receptor.

The non-DNA binding form of the rabbit uterus cytosol progesterone receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M(r) 90kDa (hsp90), a Heat shock protein Binding Immunophilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosuppressants FK506 and Rapamycin (RAP) and belongs to the FK506 binding protein family. A recombinant p59/HBI-glutathione-S-transferase fusion protein, purified by Sephadex LH-20 filtration of tritiated drug-p59/HBI complexes, binds FK506 and RAP with apparent Kd values of 75 +/- 40 and 40 +/- 15 nM, respectively. Immunopurification from cytosol of [3H]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded "9S"-PR species in which hsp90, hsp70 and p59/HBI were present. In the absence of tungstate ions, only the 4-6S PR was eluted, and Western blot analysis demonstrated the absence of hsps and p59/HBI. In contrast 30 to 50% of the original 9S-PR species containing hsps and p59/HBI, was eluted in the absence of tungstate ions but after exposure of cytosol to 5 microM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25 degrees C with 0.05 to 10 microM FK506 or RAP, then with [3H]steroids (the agonist [3H]Org 2058 or the anti-progestin [3H]RU486), contains greater amounts of 9S-PR species than that detected in non-immunosuppressant exposed control cytosol. Scatchard analysis showed an up to 2-fold decrease of the Kd value for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on immobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59/HBI associated to PR units. Altogether, these results suggest that binding of immunosuppressants to p59/HBI does not promote hsps dissociation from the receptor and, as a consequence, that inhibition of peptidyl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumption that hsp90 binds to receptor and that p59/HBI binds hsp90 but not directly to receptor, immunosuppressants affect hormone binding by an unknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the EC1 antibody specific for several mammalian p59/HBI, also binds to FK506 columns and can be eluted by exchange with either FK506 or RAP, suggesting that there is an avian HBI homolog.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Near-UV-induced radicals in Propionibacterium acnes, studied by electron spin resonance spectrometry at 77 K.

Suspensions of Propionibacterium acnes were UV irradiated and the induced radicals were measured at 77 K by electron spin resonance (ESR) spectrometry. Two types of radical were formed during irradiation and stabilized in the frozen suspensions. The relative yield of each radical was studied as a function of irradiation wavelength. The first radical, which was a singlet with a peak-to-peak width of 20 G, was insensitive to the deoxygenation of the samples and to the exchange of solvent water by heavy water. The action spectrum was similar to the absorption spectrum of NADPH. The second type of radical was not formed in deoxygenated samples and the shape of the ESR spectrum was characteristic of the superoxide radical. This radical was only formed at wavelengths below 340 nm.

Dose-Response Relationship, Radiation↗
PubMed1993

Fluorescence properties of subtilisins and related proteinases (subtilases): relation to X-ray models.

The fluorescence properties of six subtilases with known X-ray structure were determined using the same experimental conditions and instrumentation. The steady state and nanosecond lifetime measurements were performed on purified samples of phenylmethanesulphonyl-inhibited proteinases in the presence of 20 mM CaCl2 which stabilizes the molecules. The tryptophan emission quantum yield strongly depends on the local environment and varies from 0.02 to 0.10. The efficiency of tyrosine-to-tryptophan energy transfer also varies (0%-70%) in the different enzymes; the most efficient transfer was observed for thermitase. Experiments with nanosecond excitation indicated that the tryptophan fluorescence of subtilases decays with two exponential components. The X-ray models of the six proteinases were analysed in the region of the tryptophyl residues and were used to explain the observed properties.

Endopeptidases↗
PubMed2006

Instability of miRNA and cDNAs derivatives in RNA preparations.

Micro RNAs (miRNAs) are small RNA molecules, which function as important regulators of gene expression. We found that RNA preparation methods commonly utilized for miRNA expression studies yield highly unstable miRNAs. We studied the stability of four miRNAs belonging to different miRNAs families. A significant degradation of these molecules may be observed already three days after RNA isolation. Moreover, the respective cDNAs are highly unstable as well. Our findings indicate that instability of miRNAs and their cDNAs should be considered when designing miRNA expression studies.

Animals↗
PubMed2004

Mesophilic anaerobic treatment of sludge from saline fish farm effluents with biogas production.

The mesophilic anaerobic treatment of sludge from saline fish farm effluents (total solids (TS): 8.2-10.2 wt%, chemical oxygen demand (COD): 60-74 g/l, sodium (Na): 10-10.5 g/l) was carried out in continuously stirred tank reactors (CSTRs) at 35 degrees C. COD stabilization between 36% and 55% and methane yields between 0.114 and 0.184 l/g COD added were achieved. However, the process was strongly inhibited, presumably by sodium, and unstable, with propionic acid being the main compound of the volatile fatty acids (VFA). When diluting the sludge 1:1 with tap water (Na: 5.3 g/l), the inhibition could be overcome and a stable process with low VFA concentrations was achieved. The results of the study are used to make recommendations for the configuration of full-scale treatment plants for the collected sludge from one salmon farming licence and to estimate the energy production from these plants.

Anaerobiosis↗
PubMed2005

Mesophilic anaerobic treatment of sludge from salmon smolt hatching.

The mesophilic anaerobic treatment of concentrated sludge from an Atlantic salmon smolt hatchery (total solids (TS): 6.3-12.3wt%) was investigated in a continuous stirred tank reactor (CSTR) at 35 degrees C and 55-60 days hydraulic retention time (HRT). COD-stabilization between 44% and 54% and methane yields between 0.140 and 0.154l/g COD added (0.260-0.281l/g VS added) were achieved. The process was strongly inhibited, with volatile fatty acid concentrations of up to 28 g/l. But the buffer capacity was sufficient to keep the pH-value at 7.4-7.55 during the whole operation. The fertilizing value of the treated sludge was estimated to be 3.4-6.8 kg N and 1.2-2.4 kg P per ton. However, the high VFA content would necessitate special means of application. The energy from the methane that was achieved in the present study would be sufficient to cover about 2-4% of the energy demands of a flow-through hatchery.

Animals↗
PubMed2006

Nanoparticles for bioimaging.

The emergence of synthesis strategies for the fabrication of nanosized contrast agents is anticipated to lead to advancements in understanding biological processes at the molecular level in addition to progress in the development of diagnostic tools and innovative therapies. Imaging agents such as fluorescent dye-doped silica nanoparticles, quantum dots and gold nanoparticles have overcome many of the limitations of conventional contrast agents (organic dyes) such as poor photostability, low quantum yield, insufficient in vitro and in vivo stability, etc. Such particulates are now being developed for absorbance and emission in the near infrared region, which is expected to allow for real time and deep tissue imaging via optical routes. Other efforts to facilitate deep tissue imaging with pre-existing technologies have lead to the development of multimodal nanoparticles which are both optical and MRI active. The main focus of this article is to provide an overview of properties and design of contrast agents such as dye-doped silica nanoparticles, quantum dots and gold nanoparticles for non-invasive bioimaging.

Contrast Media↗
PubMed2006

Comparisons of the foaming and interfacial properties of whey protein isolate and egg white proteins.

Whipped foams (10%, w/v protein, pH 7.0) were prepared from commercially available samples of whey protein isolate (WPI) and egg white protein (EWP), and subsequently compared based on yield stress (tau(0)), overrun and drainage stability. Adsorption rates and interfacial rheological measurements at a model air/water interface were quantified via pendant drop tensiometry to better understand foaming differences among the ingredients. The highest tau(0) and resistance to drainage were observed for standard EWP, followed by EWP with added 0.1% (w/w) sodium lauryl sulfate, and then WPI. Addition of 25% (w/w) sucrose increased tau(0) and drainage resistance of the EWP-based ingredients, whereas it decreased tau(0) of WPI foams and minimally affected their drainage rates. These differing sugar effects were reflected in the interfacial rheological measurements, as sucrose addition increased the dilatational elasticity for both EWP-based ingredients, while decreasing this parameter for WPI. Previously observed relationships between tau(0) and interfacial rheology did not hold across the protein types; however, these measurements did effectively differentiate foaming behaviors within EWP-based ingredients and within WPI. Interfacial data was also collected for purified beta-lactoglobulin (beta-lg) and ovalbumin, the primary proteins of WPI and EWP, respectively. The addition of 25% (w/w) sucrose increased the dilatational elasticity for adsorbed layers of beta-lg, while minimally affecting the interfacial rheology of adsorbed ovalbumin, in contrast to the response of WPI and EWP ingredients. These experiments underscore the importance of utilizing the same materials for interfacial measurements as used for foaming experiments, if one is to properly infer interfacial information/mechanisms and relate this information to bulk foaming measurements. The effects of protein concentration and measurement time on interfacial rheology were also considered as they relate to bulk foam properties. This data should be of practical assistance to those designing aerated food products, as it has not been previously reported that sucrose addition improves the foaming characteristics of EWP-based ingredients while negatively affecting the foaming behavior of WPI, as these types of protein isolates are common to the food industry.

Animals↗
PubMed2006

An episulfide cation (thiiranium ring) trapped in the active site of HAV 3C proteinase inactivated by peptide-based ketone inhibitors.

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.

3C Viral Proteases↗
PubMed2006

One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry.

Several lines of evidence have pointed to a role of urokinase-type plasminogen activator receptor (uPAR) as a modulator of certain biochemical processes that are active during tumor invasion and metastasis. Consequently, the structure and function of this receptor have been studied extensively, using recombinantly produced uPAR that has been purified by either affinity chromatography using its cognate ligand, the urokinase-type plasminogen activator (uPA), or a monoclonal anti-uPAR antibody (R2), or by hydroxyapatite. Here, we present a new method for the efficient one-step affinity purification of recombinant uPAR exploiting a high-affinity synthetic peptide antagonist (AE152). The corresponding parent peptide was originally identified in a random phage-display library and subsequently subjected to affinity maturation by combinatorial chemistry. This study compares the affinity purification of a soluble, recombinant uPAR using the monoclonal antibody R2 or the peptide AE152 immobilized on Sepharose. The two affinity ligands perform equally well in purifying uPAR from Drosophila melanogaster Schneider 2 cell culture medium and yield products of comparable purity, activity, and stability as judged by SDS-PAGE, size exclusion chromatography and surface plasmon resonance analysis. The general availability of peptide synthesis renders the present AE152-based affinity purification of uPAR more accessible than the traditional protein-based affinity purification strategies. In this way, large amounts of recombinant uPAR can conveniently be purified for further structural and functional studies.

Amino Acid Sequence↗
PubMed1991

Hydrogen bonding in steroidogenesis: studies on new heterocyclic analogs of estrone that inhibit human estradiol 17 beta-dehydrogenase.

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.

Amino Acid Sequence↗
PubMed2003

Over-expression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript.

Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over-production. We have expressed all the genes encoding subunits of the membrane domain F(o) of the ATP synthase in a T7 RNA polymerase-based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92-171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N- and C-ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.

Bacterial Proton-Translocating ATPases↗
PubMed2001

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography.

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.

Acrylamides↗
PubMed2000

Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection.

Simple and reliable protocols are described for an extensive analysis of metabolites in extracts from different biological sources. The separation was performed by high performance ionic-exchange chromatography (HPIC) at alkaline pH using two types of chromatography columns and two detection methods. Organic acids and inorganic anions were separated on an ionPac AS11 column using a 0.5 to 35 mM Na0H gradient. Detection limits in the range of milligrams per liter were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor. Twelve phosphorylated compounds belonging to the glycolytic and the pentose phosphate pathways could be resolved on a CarboPac PA1 column using a Na0H/Na-acetate gradient. Quantification was achieved by pulsed amperometry with detection limits in the micromolar range. Cell extracts obtained by extraction in boiling buffered ethanol described previously could be directly injected onto HPIC columns for the separation of metabolites because the extraction procedure affected neither the retention time nor the stability of most of the metabolites, and yielded very clean chromatograms. These improved protocols were applied for a dynamic analysis of intracellular metabolites in Saccharomyces cerevisiae in response to a glucose pulse.

Journal Article↗