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PubMed1995

Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins.

Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside the melanosome. Their solubilization is usually carried out by treatment of melanosomal preparations with non-ionic detergents, but, so far, no comparative study of the effect of the detergents employed on the properties of the solubilized proteins has been reported. We have compared the effect of the detergents Brij-35, Nonidet P-40, Tween-20, sodium deoxycholate and Triton X-114 on several properties of the melanogenic enzymes, including the solubilization yield, stability, electrophoretic behaviour and accessibility of epitopes located in the carboxyl tail to specific antibodies. Our data indicate that not only the total amount of enzymes solubilized, but also their relative proportions in the solubilized preparations depend on the detergent used. The non-ionic detergents apparently interact strongly with the melanogenic enzymes, affecting their mobility in SDS-PAGE, and might induce different conformations of the carboxyl tail. Complete replacement of lipids by the detergents results in a decreased stability that can be partially reversed by the addition of endogenous lipids. This treatment also produces a noticeable activation of the tyrosinase isoenzymes, which is higher for TRP1 than for tyrosinase. Taken together, these data show that the transmembrane and carboxyl fragments of the proteins of the tyrosinase family might modulate the stability and activity of the melanogenic enzymes.

Animals↗
PubMed1995

Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells.

BACKGROUND: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN: Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.

Animals↗
PubMed2006

Synthesis of an estradiol glucuronide derivative and investigation of its radiopharmaceutical potential.

The aim of the current study was to synthesize a derivative of estradiol glucuronide, which is able to be labeled with 99mTc and to investigate its radiopharmaceutical potential using imaging and biodistribution studies. An estrogen derivative, beta-estradiol (1,3,5,[10]-estratriene-3,17beta-diol) attached to diethylenetriamine pentaacetic acid (DTPA) was synthesized in six steps. At the end of these steps a compound of estradiol and DTPA derivative called deoxy demethyl homoestradiolyl diethylenetriamine pentaacetic acid (ESTDTPA) was synthesized. Afterwards, this compound was reacted with UDP-glucuronyl transferase (UDPGT). Following the glucuronidation reaction, the product called deoxy demethyl homoestradiolyl diethylenetriamine pentaaceticacid-glucuronide (ESTDTPAG) was obtained. Synthesized products were purified using high performance liquid chromatography (HPLC). The identification of the purified products and impurities were also established using HPLC. Synthesized compound was labeled with 99mTc. Thin layer radio chromatography (TLRC) technique was used to determine their radiochemical yields and stabilities. Labeling yield was over 96%. The biodistribution studies were performed on female Albino Wistar rats. The activity per gram tissue was calculated and time-activity curves were plotted. The target organs (tumor, as well as uterus, ovaries, adrenals and other ER containing tissues) retain the estradiol derivative longer than nontarget organs, but even these lost most of their activity within a few hours. In addition, the imaging studies were performed on normal and tumor bearing female Albino Wistar rats using Camstar XR/T gamma camera. In gamma-scintigraphic imaging studies with 99mTc-ESTDTPAG the breast tumors could be well visualized up to 24 h.

Animals↗
PubMed2005

Theoretical proton affinities of alpha1 adrenoceptor ligands.

A systematic study has been performed of the proton affinity of a large family of agonists and antagonists of the alpha1-adrenoceptor at the B3LYP/6-31G* level of theory. After a conformational search, all the N atoms were considered as protonation sites and protonation energy values were determined. The inclusion of solvation by means of the Onsager model yielded stabilization in the proton affinity values obtained. In addition, a good correlation was found between the proton affinity values corresponding to the first protonation in gas phase of some of the compounds and their corresponding experimental affinity constants K(i) for the alpha1A adrenergic receptor.

Adrenergic alpha-Agonists↗
PubMed2004

Management of disease resistance diversity of cultivars of a species in single fields: controlling epidemics.

The use of a diversity of resistance genes limits the development of polycyclic epidemics caused by airborne pathogens and reduces the risk that resistance be overcome by virulent races. Diversity can be easily achieved by growing mixtures of cultivars with different resistance genes and homogeneous agronomic traits. The mechanisms by which disease is reduced in cultivar mixtures include the loss of inoculum due to the presence of resistant plants between susceptible ones and resistance induced by avirulent pathogens. The complementary effects of individual mixture components reacting to disease pressure and to abiotic stresses result in greater yield stability compared with pure stands. The quality of products from mixtures is at least equal to that obtained with pure stands. This type of resistance management is applicable to both annual and perennial crops.

Agriculture↗
PubMed2006

Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches.

Environmental constraints that include abiotic stress factors such as salt, drought, cold and extreme temperatures severely limit crop productivity. Improvement of crop plants with traits that confer tolerance to these stresses was practiced using traditional and modern breeding methods. Molecular breeding and genetic engineering contributed substantially to our understanding of the complexity of stress response. Mechanisms that operate signal perception, transduction and downstream regulatory factors are now being examined and an understanding of cellular pathways involved in abiotic stress responses provide valuable information on such responses. This review presents genomic-assisted methods which have helped to reveal complex regulatory networks controlling abiotic stress tolerance mechanisms by high-throughput expression profiling and gene inactivation techniques. Further, an account of stress-inducible regulatory genes which have been transferred into crop plants to enhance stress tolerance is discussed as possible modes of integrating information gained from functional genomics into knowledge-based breeding programs. In addition, we envision an integrative genomic and breeding approach to reveal developmental programs that enhance yield stability and improve grain quality under unfavorable environmental conditions of abiotic stresses.

Adaptation, Physiological↗
PubMed2006

Preparation and evaluation of nanosuspensions for enhancing the dissolution of poorly soluble drugs.

Poorly water-soluble compounds are difficult to develop as drug products using conventional formulation techniques and are frequently abandoned early in discovery. In the present study, the melt emulsification method traditionally used to prepare solid lipid nanoparticles was adapted to produce drug nanosuspensions. The method was evaluated in comparison with the well known solvent diffusion process for ibuprofen as a model drug. Control of the preparation variables (stabilizers, drug content, homogenization procedure and cooling conditions) allowed formation of nanosuspensions with diameters less than 100 nm. The major advantage of the melt emulsification method over the solvent diffusion method is the avoidance of organic solvents during production, although the mean particle size is slightly greater. The combination of Tween 80 and PVP K25 as stabilizers yields nanosuspensions with the smallest average particle size. The formulation of ibuprofen as a nanosuspension, either in the form of lyophilized powder or granules, was very successful in enhancing dissolution rate, more than 65% of the drug being dissolved in the first 10 min compared to less than 15% of the micronized drug. The increase in in vitro dissolution rate may favourably affect bioavailability and improve safety for the patient by decreasing gastric irritancy.

Calorimetry, Differential Scanning↗
PubMed2005

Synthesis and characterization of polyacrylonitrile nanoparticles by dispersion/emulsion polymerization process.

Polyacrylonitrile nanoparticles in sizes ranging from approximately 35 to 270 nm were prepared by dispersion/emulsion polymerization of acrylonitrile in a continuous aqueous phase in the presence of potassium persulfate as initiator and various alkyl-sulfate and sulfonate surfactants. The influence of various polymerization parameters (e.g., concentration of monomer and initiator, type and concentration of surfactant, temperature and time of polymerization, ionic strength, pH and co-solvent concentration) on the properties (e.g., size and size distribution, yield, stability, etc.) of the particles has been investigated. The polymerization of acrylonitrile may occur in two major locations: in the aqueous continuous phase (dispersion polymerization) and/or within the surfactant micelles (emulsion polymerization). A discussion concerning the role of these two mechanisms under different conditions, including comparison with previous literature, is also presented. Surface and bulk characterizations of the particles were performed by methods such as transmission and scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, zeta potential, and gravimetric measurements.

Acrylic Resins↗
PubMed2004

The kinetics and mechanisms of reactions of iron(III) with caffeic acid, chlorogenic acid, sinapic acid, ferulic acid and naringin.

The kinetics and mechanisms of the reactions of iron(III) with the hydroxy cinnamic acid based ligands caffeic, chlorogenic, sinapic and ferulic acids and the flavonoid naringin have been investigated in aqueous solution. The mechanisms for caffeic and chlorogenic acid are generally consistent with the formation of a 1:1 complex that subsequently decays through an electron transfer reaction. On reaction with iron(III), ferulic and sinapic acids undergo an electron transfer without the prior formation of any complex. There was no evidence of electron transfer occurring in the complex formed when iron(III) is reacted with naringin. Rate constants for k1 (formation) and k(-1) (dissociation) have been evaluated for the complex formation reactions of [Fe(H2O)6(OH)]2+ with caffeic acid, chlorogenic acid and naringin. Analysis of the kinetic data yielded stability constants, equilibrium constants for protonation of the iron(III) chlorogenic acid complex initially formed, together with the rate constants for complex decomposition through intramolecular electron transfers and in the case of caffeic acid and chlorogenic acid, rate constants for the iron(III) assisted decomposition of the initial complex formed. Some of the suggested mechanisms and calculated rate constants are validated by calculations carried out using global analysis of time dependent spectra.

Journal Article↗
PubMed2004

[Jaccoud's arthropathy. Surgical results of 41 hands].

Jaccoud's arthropathy, which was first described in 1869, is a rare syndrome that is characterized by a painless or relatively painless deformity of the digits II, II, IV and V with a dislocation of the extensor tendons into the metacarpal valley. When the thumb is affected, a Z deformity is observed. The present analysis was based on 40 patients (56 hands, 41 of which were operated on over the period 1989 to 2001). A distinction should be made between Jaccoud's disease following a known inflammatory arthropathy (23 hands; average patient age 55 years) and the idiopathic form of the disease (18 hands, average patient age 70 years). From a clinical point of view, a classification of the deformities is needed so that appropriate surgical treatment can be determined. In the present study, four groups were therefore proposed, in groups I and III the realignment to centre of the extensor tendons of the metacarpophalangeal joint and joint stabilization yielded 83% positive results. In groups II and IV the results were only 66% good after Swanson implant. The pathogenesis of Jaccoud's disease has not yet been determined, but now that more information has become available and a better analysis can be made of its various clinical and anatomopathological charasteristics, these should assist in defining precise surgical indications.

Aged↗
PubMed2026

A cis-regulatory allele of ZmNPR1I spatially uncouples flowering from stalk-rot resistance in maize.

Pleiotropic effects of adaptive genes frequently constrain crop improvement by coupling beneficial traits with unfavorable trade-offs. In maize, ZmCCT10 confers strong stalk-rot resistance but causes delayed flowering under long-day conditions, limiting its deployment in temperate breeding. Here, we identify qPss3 as a cis-regulatory locus that governs its downstream target gene, ZmNPR1I. The ZmNPR1I protein represses ZmCCT10 transcription and, together with ZmNPR1-3, facilitates ZmCCT10 protein degradation. The favorable qPss3A5 allele reduces ZmNPR1I expression in leaves, relieving repression of ZmCCT10, ZmSPL32, and ID1, which collectively enhance ZCN8 expression to accelerate flowering. In roots, however, pathogen-induced activation of the resistant ZmCCT10H5 allele largely bypasses qPss3 regulation, thereby preserving stalk-rot resistance. Introgression of qPss3A5 into ZmCCT10H5-containing maize germplasm restores flowering adaptation without compromising disease resistance, improving yield stability under disease pressure. Our work reveals a tissue-specific qPss3A5-ZmNPR1I regulatory module that uncouples the developmental and immune functions of a pleiotropic adaptive gene, providing a general strategy for optimizing beneficial alleles in crop breeding.

flowering time↗
PubMed2005

High-concentration food wastewater treatment by an anaerobic membrane bioreactor.

The viability of treating high-concentration food wastewater by an anaerobic membrane bioreactor (AMBR) was studied using polyethersulfone (PES) ultrafiltration membranes PES200, PES300, PES500 and PES700 with norminal molecular weight cutoff (MWCO) ranging from 20,000 to 70,000 Da. Hydraulic and solid retention time significantly affected the treatment performance of the AMBR kept at 60 h and 50 days in the study. The four membranes exhibited a similar efficiency in removal of suspended solids, color, chemical oxygen demand (COD) and bacteria. When the volumetric loading rate was below 4.5 kg/m3d, COD removal rate was in the range of 81-94% and the gas yield stabilized at 0.136 m3/kg COD. The effect of membrane properties including MWCO, hydrophobicity and surface morphology on membrane fouling and cleaning was evaluated. The PES200 membranes with the smallest MWCO and smoothest surface exhibited a serious initial flux decline, whereas the PES700 membranes with the largest MWCO and roughest surface were observed related to the highest flux decline and the lowest recoverable flux rate during long-term operation. Membrane autopsy revealed that the significant flux decline was caused by the formation of a thick biofouling layer onto the membrane surfaces.

Anaerobiosis↗
PubMed2003

Conformation of reconstituted mononucleosomes and effect of linker histone H1 binding studied by scanning force microscopy.

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.

Air↗
PubMed1990

Selection of electrophoric derivatives of 1-aminopyrene and 2-aminofluorene for determination by gas chromatography with electron-capture negative-ion mass spectrometry.

Several electrophoric derivatives of 1-aminopyrene and 2-aminofluorene were prepared. Reagents such as heptafluorobutyryl chloride, pentafluorobenzoyl chloride, pentafluorobenzyl bromide and pentafluorobenzaldehyde, alone and in certain combinations, were employed. The ease of formation, yield, stability and fragmentation by gas chromatography with electron-capture negative-ion (ECNI) mass spectrometry of the derivatives were compared. This allowed the most promising ones to be selected for future work on the sensitive detection of aminopolyaromatics by this detection technique. Pentafluorobenzylidene (first choice) and N-pentafluorobenzyl-N-heptafluorobutyryl (second choice) derivatives emerged as the best ones. The origins of losses of HF and 2HF from some of the derivatives were elucidated in the ECNI mass spectra by studies of deuterium-labeled analogues.

Chemistry Techniques, Analytical↗
PubMed1989

Beyond 1 year after liver transplantation.

We retrospectively reviewed the long-term results in 46 patients who survived at least 1 year after liver transplantation. Only one death has occurred, and one patient has required retransplantation. Biochemical liver function tests showed median values in the normal range, except for mild elevation of serum gamma-glutamyltransferase. In patients with primary biliary cirrhosis, these test results were completely normal. A liver biopsy 1 year after transplantation disclosed normal histologic findings in 31 patients (67%). The other patients had either transient (acute rejection) or stable (chronic rejection) abnormalities, except for two patients with progressive graft dysfunction attributable to chronic rejection. A clinically significant vascular anastomotic abnormality was noted in one patient who had hepatic artery thrombosis. Late bile duct complications occurred in 15% of patients, all of whom had a satisfactory outcome after surgical or radiologic intervention. Cyclosporine-related nephrotoxicity and hypertension each occurred in 67% of patients; however, conversion to a low-dose cyclosporine-azathioprine regimen yielded stabilization of renal function after the first postoperative year, and hypertension has been easily controlled medically. Diabetes necessitating insulin treatment developed in three patients. The body weight of the study patients had increased by a median of 6.5 kg at 1 year but stabilized thereafter. Subjective well-being and satisfaction with life were reported by 91% of the patients. Of the 46 patients, 26 were employed, 16 were homemakers, and only 4 did not work, 2 because of transplant-related medical problems. Thus, we conclude that liver transplantation rehabilitates patients with end-stage liver disease and enhances their quality of life.

Adolescent↗
PubMed2001

Effect of temperature increase from 55 to 65 degrees C on performance and microbial population dynamics of an anaerobic reactor treating cattle manure.

The effect of a temperature increase from 55 to 65 degrees C on process performance and microbial population dynamics were investigated in thermophilic, lab-scale, continuously stirred tank reactors. The reactors had a working volume of 31 and were fed with cattle manure at an organic loading rate of 3 g VS/l reactor volume/d. The hydraulic retention time in the reactors was 15 days. A stable reactor performance was obtained for periods of three retention times both at 55 degrees C and 65 degrees C. At 65 degrees C methane yield stabilized at approximately 165ml/g VS/d compared to 200 ml/g VS/d at 55 degrees C. Simultaneously, the level of total volatile fatty acids, VFA, increased from being below 0.3 g/l to 1.8-2.4 g acetate/l. The specific methanogenic activities (SMA) of biomass from the reactors were measured with acetate, propionate, butyrate, hydrogen, formate and glucose. At 65 degrees C. a decreased activity was found for glucose-, acetate-, butyrate- and formate-utilizers and no significant activity was measured with propionate. Only the hydrogen-consuming methanogens showed an enhanced activity at 65 degrees C. Numbers of cultivable methanogens, estimated by the most probable number (MPN) method, were significantly lower on glucose, acetate and butyrate at the increased operational temperature, while the numbers of hydrogenotrophic methanogens remained unchanged. No viable propionate-degrading bacteria were enriched at 65 C. Use of ribosomal oligonucleotide probes showed that an increase in temperature resulted in a decreased contribution of the rRNA of the domain bacteria from 74-79 to 57-62% of the universal probe, while the rRNA of the domain archaea, increased from 18-23 to 34-36%.

Anaerobiosis↗
PubMed2000

[Jaccoud's arthropathy].

Jaccoud's arthropathy, which was first described in 1869, is a rare syndrome that is characterized by a painless or relatively painless deformity of the digits II, II, IV and V with a dislocation of the extensor tendons in the metacarpal fossae. When the thumb is affected, a Z-deformity is observed. The present analysis was based on 21 patients (30 hands, 24 of which were operated on over the period 1989 to 1996). A distinction should be made between Jaccoud's disease following a known inflammatory arthropathy (15 hands; average patient age 55 years) and the idiopathic form of the disease (9 hands; average patient age 70 years). From a clinical point of view, a classification of the deformities is needed so that appropriate surgical treatment can be determined. In the present study, four groups were therefore proposed: in groups I and II, the realignment to centre of the extensor tendons over the metacarpophalangeal joint and joint stabilization yielded 87% positive results. In groups III and IV, the results were not as satisfactory either for the Swanson implant or for metacarpal shortening osteotomy. The pathogenesis of Jaccoud's disease has not yet been determined, but now that more information has become available and a better analysis can be made of its various clinical and anatomopathological characteristics, these should assist in defining precise surgical indications.

Aged↗
PubMed1976

Nuclear magnetic resonance determination of intramolecular distances in bovine pancreatic trypsin inhibitor using nitrotyrosine chelation of lanthanides.

Nitration of tyrosine has been investigated as a means for chemically introducing lanthanide chelating sites at known positions in proteins. The low-field portions of the 250-MHZ and 270-MHZ 1H nuclear magnetic resonance spectra of native and chemically modified bovine pancreatic trypsin inhibitor have been studied in the presence of lanthanide ions. Comparisons of spectral changes observed with native, mononitro (tryosine 10) and dinitro (tyrosines 10 and 21) derivatives enable these changes to be separately attributed to metal bound at nitrotyrosine 21, nitrotyrosine 10, or the set of five carboxyl groups. The pH dependence of Pr(III) and Eu(III) induced chemical shifts yields stability constants of 50 and 159 M-1 for the association between lanthanides and nitrotyrosines 10 and 21, respectively. Correlation times for the interactions with Gd(III) bound to specific nitrotyrosines are estimated from the induced line broadening of resonances of the nitrotyrosine ring protons. These stability constants and correlation times are used to determine the distances from the different metal binding sites to buried backbone NH protons having resolved resonances. Comparisons with distances in the x-ray crystal structure give assignments of the NH resonances to a small set of buried backbone NH's.

Animals↗