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At least 1,675 records · Page 93Linked to original sources
PubMed2005

Quantitative magnetization transfer by trains of radio frequency pulses in human brain: extension of a free evolution model to continuous-wave-like conditions.

A theoretical model of free evolution between repeated magnetic transfer (MT) pulses was extended to continuous-wave (CW)-like conditions showing that only the repetitive "direct" saturation of bulk water changes the transient and stationary behavior. The influence of the pulse repetition period (PR) on progressive saturation was studied in cortical gray matter (GM) and central white matter (WM) under conditions of short periods of free evolution and strong macromolecular saturation. Interpulse delays of 3 ms were achieved in vivo on a 1.5-T MR system with bell-shaped MT pulses of 12-ms duration and nominal flip angles of up to 1440 degrees and single-shot readout by a stimulated echo acquisition mode localization sequence. The frequency offset was chosen between 1 and 3 kHz to avoid excessive direct saturation. The stationary MT ratio (MTR) followed an inverse linear PR dependence, showing a consistent partial saturation of about 90% at zero PR for both WM and GM. Comparison to a relaxation-matched liquid indicated the presence of MT, but not necessarily of direct saturation. The transient behavior indicated considerable direct saturation, but this could also be explained by MT. These inconsistencies showed that the intervals of time evolution in our experiments were too long to be modeled by CW-like conditions. Free evolution takes place during the whole PR rather than during the interpulse delay only. Quantification using the rates of free evolution theory yielded the saturations and rate constants necessary to explain the observed behavior. The theory of rapid CW-like pulsing provides an upper limit for the rate of progressive saturation. This limit is approached at PR below an estimated value of 5 ms. The phenomenological PR dependence of the steady-state MTR may indicate that MT exceeded the direct saturation. Unlike to an idealized CW experiment, the extrapolated value at zero PR is subject to direct effects and not a physically meaningful constant.

Adult↗
PubMed2003

The shape parameter of liposomes and DNA-lipid complexes determined by viscometry utilizing small sample volumes.

A minicapillary viscometer utilizing <0.5 ml of sample at a volume fraction of <0.1% is described. The calculated a/b of DPPC/DPPG multilamellar liposome was 1.14 as prolate ellipsoids and a/b of dioleoylpropyltrimethyl ammonium methylsulfate-DNA complex at a charge ratio of 4:1 (+/-) was 3.7 as prolate ellipsoids or 4.9 as oblate ellipsoids. The deviation of shape from perfect sphere is thus expressed quantitatively in more than two significant figures. In these measurement, the necessary amount of DNA is <0.5 mg.

Capillary Action↗
PubMed2003

Downregulation of IRS-1 protein in thapsigargin-treated human prostate epithelial cells.

Thapsigargin treatment of cultured cells leads to an increase in the intracellular calcium concentration, activation of calpain, and, in some cell types, apoptosis. Using a human prostate epithelial cell line that undergoes apoptosis in the presence of thapsigargin, we find decreased levels of IRS-1 protein levels during apoptosis. Inhibition of calpain prevents this decrease in IRS-1 protein; however, inhibitors of caspases or the proteasome are ineffective in maintaining IRS-1 levels. In terms of IGF-I-related second messenger proteins, the effect of thapsigargin is specific for IRS-1 since the protein levels of IGF-I receptor beta-subunit, Akt, Erk, and Shc are not affected. In addition to preventing the reduction in IRS-1, treatment of cells with calpain inhibitor II prevents apoptosis in response to thapsigargin. Finally, IRS-1 and calpain can be identified in protein complexes isolated using IRS-1-specific antibodies, indicating that calpain can associate with either IRS-1 or one of the proteins present in protein complexes that contain IRS-1. In total, these results suggest that IRS-1 may be targeted for degradation by calpain during apoptosis.

Apoptosis↗
PubMed1999

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.

Adenosine Triphosphatases↗
PubMedDate not supplied

alpha-Crystallin quaternary structure and interactive properties control eye lens transparency.

The eye lens is the foremost biological system where function is directly under control of the physico-chemical properties of the cytoplasmic macromolecular solution. Indeed, lens transparency and opacity, lens refractive index gradient and viscosity, are the result of the structural and interactive properties of the crystallins, of their stability, of the fine tuning of their interaction potentials and associations at different levels of organization. Among the different crystallin classes, alpha-crystallins have represented a major challenge for a long time. The alpha-crystallin secondary, tertiary and quaternary structures are still unknown. On the functional side, however, it is established that alpha-crystallin quaternary structure and repulsive interactions determine lens transparency, whereas the alpha-crystallin chaperone effect most probably plays a role in the aging process. In the present paper, we recall the physico-chemical properties and the quaternary structure features of alpha-crystallins that were demonstrated to control light scattering and transparency. The interest of a crystallin mixture for lens function is discussed. Then, a formal approach is proposed to design models for the alpha-crystallin quaternary structure, including the question of whether alpha-crystallins assemble with symmetry. An hypothesis relevant to the fold of the alpha-crystallin C-terminal domain is presented in another paper in this issue.

Animals↗
PubMed2002

Stereocomplexes based on poly(lactic acid) and insulin: formulation and release studies.

Diastereoisomeric complexes of insulin with D-poly(lactic acid) (D-PLA) or stereocomplexes of D- and L-PLA entrapping insulin were discovered. The complexes were spontaneously formed when insulin and D-PLA were mixed together in acetonitrile solution. Complexes of insulin-D-PLA formed a microparticulate precipitate after a few hours in the solution. The porous 1-3 microm precipitate, which contained both insulin and D-PLA, was insoluble in solvents that dissolve isotactic PLA, and had an additional transition temperature at 169 degrees C. When suspending these particles in buffer solution of pH 7.4, 37 degrees C, insulin was constantly released for a few weeks. L-PLA or D,L-PLA did not form a precipitate with insulin, which indicates stereospecificity to the complex formation. Microparticulates were also obtained when L-PLA was added to the D-PLA-insulin solution. In this case two types of complexes, D-PLA-insulin and D-PLA-L-PLA complexes, were formed. These macromolecular stereocomplexes may form the onset of the development of a new generation of controlled release systems for peptides and proteins, by molecular complexation with enantiomeric polymers.

Acetonitriles↗
PubMed2004

Hyaluronan microspheres for sustained gene delivery and site-specific targeting.

Hyaluronan is a naturally occurring polymer that has enjoyed wide successes in biomedical and cosmetic applications as coatings, matrices, and hydrogels. For controlled delivery applications, formulating native hyaluronan into microspheres could be advantageous but has been difficult to process unless organic solvents are used or hyaluronan has been modified by etherification. Therefore, we present a novel method of preparing hyaluronan microspheres using adipic dihydrazide mediated crosslinking chemistry. To evaluate their potential for medical applications, hyaluronan microspheres are incorporated with DNA for gene delivery or conjugated with an antigen for cell-specific targeting. The results show that our method, originally developed for preparing hyaluronan hydrogels, generates robust microspheres with a size distribution of 5-20mum. The release of the encapsulated plasmid DNA can be sustained for months and is capable of transfection in vitro and in vivo. Hyaluronan microspheres, conjugated with monoclonal antibodies to E- and P-selectin, demonstrate selective binding to cells expressing these receptors. In conclusion, we have developed a novel microsphere preparation using native hyaluronan that delivers DNA at a controlled rate and adaptable for site-specific targeting.

Animals↗
PubMedDate not supplied

The insulin receptor kinase.

The insulin receptor appears as a tetrameric glycoprotein consisting of two Mr 130,000 subunits (alpha), and two Mr 95,000 subunits (beta) in a disulfide-linked complex. Insulin bound to its specific cell surface receptors in its target cells leads to a complex array of molecular events resulting in insulin effects. It is now generally believed that protein phosphorylation-dephosphorylation reactions represent an important mechanism by which a variety of extracellular stimuli regulate cellular functions. Insulin mediates such reactions, but it is not known whether these are the biochemical link between the binding of insulin to its receptor and its final cellular effects. In search of initial post-binding events which might play a role in insulin action, we looked for phosphorylation of insulin receptors. We show that the insulin receptor displays two functional domains, an insulin binding alpha-subunit, and an insulin responsive protein kinase contained in the beta-subunit. We envisage the insulin receptor as an integrated system for transmembrane signal transmission in which hormone binding to the alpha-subunit leads to activation of the beta-subunit via conformational changes.

Adenosine Triphosphate↗
PubMed2002

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization.

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.

Action Potentials↗
PubMed1998

Reconstitution of human telomerase activity in vitro.

Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2. Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.

Carrier Proteins↗
PubMed1995

750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma.

High-resolution 750 MHz 1H NMR spectra of control human blood plasma have been measured and assigned by the concerted use of a range of spin-echo, two-dimensional J-resolved, and homonuclear and heteronuclear (1H-13C) correlation methods. The increased spectral dispersion and sensitivity at 750 MHz enable the assignment of numerous 1H and 13C resonances from many molecular species that cannot be detected at lower frequencies. This work presents the most comprehensive assignment of the 1H NMR spectra of blood plasma yet achieved and includes the assignment of signals from 43 low M(r) metabolites, including many with complex or strongly coupled spin systems. New assignments are also provided from the 1H and 13C NMR signals from several important macromolecular species in whole blood plasma, i.e., very-low-density, low-density, and high-density lipoproteins, albumin, and alpha 1-acid glycoprotein. The temperature dependence of the one-dimensional and spin-echo 750 MHz 1H NMR spectra of plasma was investigated over the range 292-310 K. The 1H NMR signals from the fatty acyl side chains of the lipoproteins increased substantially with temperature (hence also molecular mobility), with a disproportionate increase from lipids in low-density lipoprotein. Two-dimensional 1H-13C heteronuclear multiple quantum coherence spectroscopy at 292 and 310 K allowed both the direct detection of cholesterol and choline species bound in high-density lipoprotein and the assignment of their signals and confirmed the assignment of most of the lipoprotein resonances.

Adult↗
PubMed1994

Prolyl 4-hydroxylase: defective assembly of alpha-subunit mutants indicates that assembled alpha-subunits are intramolecularly disulfide bonded.

The vital hydroxylation of peptidyl proline residues in collagens and protein with collagen-like amino acid sequences is catalyzed by the tetrameric enzyme prolyl 4-hydroxylase (P4-H). We have previously detailed [John et al. (1993) EMBO J. 12, 1587-1595] the redox-dependent assembly of the catalytically important alpha-subunit (64 kDa) in a cell-free system containing endogenous beta-subunits (PDI, 60 kDa). To identify the origin of this redox-dependent assembly, we have now shown directly by an electrophoretic mobility shift assay that the assembled wild-type protein possesses at least one intramolecular disulfide bond. We also analyzed five alpha-subunit mutants that have single Cys to Ser mutations in one of the five Cys residues present in the wild-type protein and found that (i) subunits mutated at Cys150 or Cys511 formed intramolecular disulfide bonds, whereas subunits mutated at Cys276, Cys293, or Cys486 did not, (ii) mutation of Cys276, Cys293, or Cys486 led to a large reduction in alpha-beta complex formation, (iii) subunits mutated at Cys276, Cys293, Cys486, or Cys511 were recognized by an antiserum raised against an alpha-subunit C-terminal peptide which failed to recognize the assembled wild-type subunit or the assembled subunit mutated at Cys150, and (iv) the assembled complexes fractionated in a similar position to the purified protein on sucrose gradients whereas the assembly-defective mutants formed higher molecular weight aggregates or complexes with other proteins. On the basis of these results, we propose that P4-H alpha-subunits possess an intramolecular disulfide bond between Cys276 and Cys293 that is essential for alpha-beta complex formation.

Animals↗
PubMed1985

Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.

The purified gamma subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of gamma was accomplished by separating the disulfide-linked alpha-gamma subunit from the noncovalently associated beta chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified alpha and gamma exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrations and in the presence of excess serum albumin. Purified gamma also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only alpha and beta. These results indicate that alpha possesses a specific site for interaction with gamma and that this site is preserved in the isolated subunit. Furthermore, this site remains accessible when alpha is associated with beta. In related experiments, gamma was found to specifically associate with membrane-bound C5b-8' and C5b-(8')9 complexes. These results indicate that the site for gamma interaction remains accessible on alpha in C5b-8' and is not shielded by C9 within C5b-(8')9. It is concluded that the gamma subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9.

Binding Sites↗
PubMed1986

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR.

Sequence-specific resonance assignments of the 1H NMR spectrum of the 85-residue histidine-containing phosphocarrier protein (HPr) are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. Additional side-chain assignments have been made with long-range coherence transfer experiments [Klevit, R. E., & Drobny, G. P. (1986) Biochemistry (second paper of three in this issue)]. In this paper, the NMR assignments were used to determine the secondary structure and the tertiary folding of HPr in solution. The secondary structural elements of the protein were determined by visual inspection of the pattern of nearest-neighbor nuclear Overhauser effects (NOEs) and the presence of persistent amide resonances. Escherichia coli HPr consists of four beta-strands, three alpha-helices, four reverse turns, and several regions of extended backbone structure. Long-range NOEs, especially among side-chain protons, were used to determine the tertiary structure of the protein by use of the secondary structural components. The four beta-strands form a single antiparallel beta-pleated sheet. The hydrophobic faces of the alpha-helices interact to form a hydrophobic core and sit above the hydrophobic face of the beta-sheet, forming an open-face beta-sheet sandwich structure. The active site histidine, His-15, is on a short kinked segment of backbone that is accessible to the solvent. The positively charged phosphorylation site (His-15 and Arg-17) interacts with the negatively charged carboxyl terminus of the protein (Glu-85).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands.

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.

Bacillus megaterium↗
PubMedDate not supplied

Methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) copolymer micelles for formulation of hydrophobic drugs.

Six amphiphilic diblock copolymers based on methoxy poly(ethylene glycol) (MePEG) and poly(delta-valerolactone) (PVL) with varying hydrophilic and hydrophobic block lengths were synthesized via a metal-free cationic polymerization method. MePEG-b-PVL copolymers were synthesized using MePEG with Mn = 2000 or Mn = 5000 as the macroinitiator. 1H NMR and GPC analyses confirmed the synthesis of diblock copolymers with relatively narrow molecular weight distributions (Mn/Mw = 1.05-1.14). DSC analysis revealed that the melting temperatures (Tm) of the copolymers (47-58 degrees C) approach the Tm of MePEG as the PVL content is decreased. MePEG-b-PVL copolymer aggregates loaded with the hydrophobic anti-cancer drug paclitaxel were found to have effective mean diameters ranging from 31 to 970 nm depending on the composition of the copolymers. A MePEG-b-PVL copolymer of a specific composition was found to form drug-loaded micelles of 31 nm in diameter with a narrow size distribution and improve the apparent aqueous solubility of paclitaxel by more than 9000-fold. The biological activity of paclitaxel formulated in the MePEG-b-PVL micelles was confirmed in human MCF-7 breast and A2780 ovarian cancer cells. Furthermore, the biocompatibility of the copolymers was established in CHO-K1 fibroblast cells using a cell viability assay. The in vitro hydrolytic and enzymatic degradation of the micelles was also evaluated over a period of one month. The present study indicates that the MePEG-b-PVL copolymers are suitable biomaterials for hydrophobic drug formulation and delivery.

Animals↗
PubMed1991

A comparative NMR study between the macrolide antibiotic roxithromycin and erythromycin A with different biological properties.

1H nuclear Overhauser enhancement studies and 1H NMR 3J analysis establish the similarity between the major solution-state conformation of roxithromycin (1) and the erythromycin (2). A major difference between the structure of antibiotics 1 and 2 is the replacement of the 9-keto group in 2 by a 9-[O-(2,5-dioxahexyl)oxime] group. The NOE studies show that this oxime chain is oriented above the macrocyclic lactone ring and that the oxygen atoms of this chain are engaged in tight hydrogen bonding with a water molecule and with the 6- and 11-hydroxyl groups of the macrocycle. It results in a globular form of the whole roxithromycin molecule. These data explain also a relative hydrophobicity of this antibiotic. Erythromycin A (2), which presents a less rigid macrocycle with two free hydroxyl groups (6-OH and 11-OH), forms a dimer detected by FAB mass spectroscopy. 1H and 13C NMR relaxation measurements (T1) for both antibiotics show that interresidue hydrogen bonds in roxithromycin reduce the rotational freedom of the macrocyclic lactone ring and consequently the motions of desosamine and cladinose sugars. In another way, an ionization of the amino function occurs in the various media according to the nature of the antibiotic. This would allow the reactivity modification of the desosamine unit. In the biological study, the modifications of the 455-nm metabolite-cytochrome P-450 complex formation are observed.

Crystallization↗