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PubMed2004

Lower frequency of CYP2C9*2 in Mexican-Americans compared to Spaniards.

Interethnic differences in cytochrome P450 polymorphism might be responsible, at least in part, for the variations in drug disposition between ethnic groups. Of the various CYP2C9 alleles, CYP2C9*2 and CYP2C9*3 have been reported to have altered catalytic activities compared to the wild-type CYP2C9*1. The present study is aimed at analysing the CYP2C9 polymorphism in a Mexican-American compared with a Spanish population. Differences between the two populations of healthy volunteers, Mexican-Americans (n=98 subjects) and Spaniards (n=102 subjects), regarding the CYP2C9 allele frequencies have been found. CYP2C9 genotypes among the studied Mexican-American population are in equilibrium. The 95% CI were, respectively, 0.81-0.90 for CYP2C9*1 (n=169), 0.05-0.13 for CYP2C9*2 (n=16) and 0.031-0.10 for CYP2C9*3 (n=11). CYP2C9*4, *5 and *6 were found in none of the studied subjects. The frequency of CYP2C9*2 was lower among Mexican-Americans compared to Spaniards (P<0.05). The obtained frequency of CYP2C9 alleles is compatible with the genomic assembly of the constitutive potential ethnic origin of this population, and supports the need of pharmacogenetic studies for optimizing the recommended drug dosages to Mexican-Americans.

Adult↗
PubMed2006

Conical and glancing Jahn-Teller intersections in the cyclic trinitrogen cation.

The ground and electronically excited states of cyclic N(3) (+) are characterized at the equilibrium D(3h) geometry and along the Jahn-Teller distortions. Lowest excited states are derived from single excitations from the doubly degenerate highest occupied molecular orbitals (HOMOs) to the doubly degenerate lowest unoccupied molecular orbitals (LUMOs), which give rise to two exactly and two nearly degenerate states. The interaction of two degenerate states with two other states eliminates linear terms and results in a glancing rather than conical Jahn-Teller intersection. HOMO-2-->LUMOs excitations give rise to two regular Jahn-Teller states. Optimized structures, vertical and adiabatic excitation energies, frequencies, and ionization potential (IP) are presented. IP is estimated to be 10.595 eV, in agreement with recent experiments.

Journal Article↗
PubMed1995

Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo.

Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.

Bacterial Proteins↗
PubMed1984

A monoclonal antibody to human blood group B. Performance, evaluation and optimisation.

A monoclonal anti-B antibody (ES-4) was obtained by fusing spleen cells from a mouse immunized with soluble human blood group B substance with mouse myeloma cell line NS-1. The antibody was shown to agglutinate optimally group B cells at pH 7.2 and 0.15 ionic strength. Increasing the ionic strength to 0.24 gave optimal reactivity over a wider pH range. Culture supernatant containing anti-B (ES-4), after pH and ionic strength adjustment could be used in unconcentrated form as a red cell typing reagent. Anti-B (ES-4) agglutinated five examples of cells of the Bweak (Bw) phenotype and one example of acquired B phenotype. In contrast three of the Bw cells and the acquired B phenotype were not agglutinated by a commercial monoclonal anti-B by a tube technique. The data suggested that the equilibrium constant of the monoclonal anti-B (ES-4) was higher than that of the commercial reagent.

ABO Blood-Group System↗
PubMed2004

Discovery of picomolar slow tight-binding inhibitors of alpha-fucosidase.

Glycosidase inhibitors have shown great medicinal and pharmaceutical values as exemplified by the therapeutic treatment of influenza virus and non-insulin-dependent diabetes. We herein report the discovery of picomolar slow tight-binding inhibitors 2-5 against the alpha-fucosidase from Corynebacterium sp. by a rapid screening for an optimal aglycon attached to 1-aminomethyl fuconojirimycin (1). The time-dependent inhibition displays the progressive tightening of enzyme-inhibitor complex from a low nanomolar K(i) to picomolar K(i)* value. Particularly compound 2 with a K(i)* of 0.46 pM represents the most potent glycosidase inhibitor to date. The effect of compound 3 on the intrinsic fluorescence of alpha-fucosidase is both time- and concentration-dependent in a saturation-type manner, which is consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (E.I), followed by the slower formation of a tightly bound enzyme-inhibitor complex (E.I*). The binding affinity increases 3.5 x 10(4)-fold from 1 (K(i) = 16.3 nM) to 2 (K(i)* = 0.46 pM). This work clearly demonstrates the effectiveness of our combinatorial approach leading to the rapid discovery of potent inhibitors.

1-Deoxynojirimycin↗
PubMed2005

Synthesis and in vitro examination of [124I]-, [125I]- and [131I]-2-(4-iodophenylamino) pyrido[2,3-d]pyrimidin-7-one radiolabeled Abl kinase inhibitors.

The pyridopyrimidinones are a potent class of inhibitors of c-Abl kinase and Bcr-Abl kinase, the causative fusion protein in chronic myelogenous leukemia and Src family kinases. A novel method for routine, high-yield no-carrier-added synthesis of [(124)I]-, [(125)I]- and [(131)I]-6-(2,6-dichlorophenyl)-2-(4-iodophenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one has been developed. The 4'-trimethylstannyl- or 4'-tri-n-butylstannyl-pyridopyrimidinone precursors were prepared from the aryl bromide via a palladium-mediated coupling with hexaalkylditin (dioxane/microwave irradiation/10 min at 160 degrees C). The radioiodination of 4'-stannylpyridopyrimidinones was found to optimally occur via an iododestannylation with Na(124)I, Na(125)I or Na(131)I in the presence of an oxidant [30% H(2)O(2)/HOAc (1:3)/10 min] in 79-87% radiochemical yield with >99% radiochemical purity. The total radiosynthesis time was 30 min. The 4-iodophenylpyridopyrimidinone 2 inhibited recombinant Abl kinase activity with an IC(50) of 2.0 nM. Cell proliferation of K562 and A431 cells was inhibited with an IC(50) of 2.0 and 20 nM, respectively. Rapid cellular uptake and equilibrium were observed within 10-15 min using [(131)I]-4-iodophenylpyridopyrimidinone 6c in K562 and A431 cells and demonstrated a 2.8-fold uptake selectivity for the Bcr-Abl-expressing K562 cells at 60 min. These results suggest that pyridopyrimidinone radiotracers may be useful in imaging Abl-, Bcr-Abl- or Src-expressing malignancies.

Carcinoma, Squamous Cell↗
PubMed1983

Suspension culture of human mast cells/basophils from umbilical cord blood mononuclear cells.

Selective growth of human mast cells/basophils was obtained in suspension cultures of mononuclear cells from umbilical cord blood. A fraction of culture supernatant of phytohemagglutinin-stimulated T cells, which lacked interleukin 2, was required for the selective growth of mast cells. When the mononuclear cells were cultured for 2-4 wk in the presence of the fraction, 50-90% of the total cells in the cultures contained metachromatic granules. Under the optimal culture conditions, the number of mast cells/basophils recovered from the cultures was 30-60% of the number of mononuclear cells plated. Cultured mast cells/basophils bear 1.2-3.83 X 10(5) IgE receptors per cell and contained 0.48-1.6 micrograms of histamine per 10(6) cells. The average forward rate constant, k1, and dissociation constant, k-1, for the binding of human IgE to IgE receptors on the cells were 1.9 X 10(5) M-1 sec-1 and 6.9 X 10(-5) sec-1, respectively (average equilibrium constant = 2.75 X 10(9) M-1). Specific binding of human IgE with high affinity indicates that the cells recovered in the suspension culture are human mast cells/basophils. Cultured cells sensitized with human IgE released a substantial amount of histamine upon exposure to anti-IgE. The results indicate that human mast cells/basophils obtained in the culture are functionally mature.

Basophils↗
PubMed1998

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.

Humans↗
PubMed1986

Beneficial effect of glycaemic improvement in non-ischaemic forms of diabetic retinopathy: a 3-year follow-up.

Recent studies have suggested that optimal glucose control fails to arrest or even worsens background retinopathy possibly by aggravating retinal ischaemia. Fourteen insulin-dependent diabetic patients, aged 34 +/- 11 years (mean +/- SD) treated by long-term intraperitoneal insulin infusion using portable pumps, were followed for 3 years. Preproliferative or proliferative lesions on ophthalmoscopy and large non-perfused areas on fluorescein angiography were exclusion criteria. Six patients were found to have minimal and 8 mild background retinopathy. The patients were retrospectively assigned to two comparable groups except for their glycaemic equilibrium under insulin infusion: average control (n = 6) and excellent control (n = 8), although glycaemic control was significantly improved in all cases when compared to previous conventional therapy. Fluorescein changes were scored blindly and independently by 3 ophthalmologists according to modifications of fluorescein diffusion and capillary abnormalities. The two types of retinal lesions improved gradually in 10 patients and deteriorated in only 1 patient, although not progressing to proliferative retinopathy. Structural improvements were significantly more frequent in the excellently controlled group. We conclude that non-ischaemic lesions may still be arrested and even improved by tight metabolic control.

Adult↗
PubMed1997

Time-dependent intracellular accumulation of delta-aminolevulinic acid, induction of porphyrin synthesis and subsequent phototoxicity.

Photodynamic therapy (PDT), a novel treatment for a variety of human malignancies, usually consists of visible light irradiation of lesions following the systemic administration of a photosensitizer. Induction of the endogenous photosensitizer protoporphyrin IX by the systemic or topical administration of delta-aminolevulinic acid (delta-ALA) is being investigated for use in PDT. We have determined that the incubation of two human and two rodent tumor cell lines in culture with delta-ALA over a 24 h period results in an increase in the accumulation of fluorescent porphyrins in all of these cell lines. However, the two human cell lines produce fluorescent porphyrin at different rates from those seen in the rodent cell lines. The uptake of 14C-delta-ALA was concentration dependent, similar for all the cell lines studied and rapidly reached an intra/extracellular equilibrium after delta-ALA was added to the culture medium. The increase in intracellular fluorescent porphyrin was dependent on the level of delta-ALA in the medium and the incubation time and was directly related to the phototoxicity observed upon exposure of cultured monolayers to light. The data demonstrate that equivalent levels of phototoxicity can be attained by exposing cells to 0.04 mM delta-ALA for 24 h or to 0.5 mM delta-ALA for 2 h. These findings may have implications for optimization of PDT treatment regimens that use delta-ALA.

Aminolevulinic Acid↗
PubMed1981

Application of the law of mass action to design and performance of receptor assays for 1,25(OH)2 vitamin D.

The binding of 1,25(OH)2 vitamin D3 to its chick duodenal cytosol receptor protein has been analysed on a law of mass action model. Within the limits of experimental error there is such good agreement, when only one binding site is considered, that it is possible to fit the standard assay curve for practical use. Simple pocket-calculator methods are given for this. The equilibrium constant for the reaction, under the conditions used (pH 7.4, 25 degrees C), was 2.1 X 10(10) 1 X mol-1. A kinetic analysis under the same conditions yielded forward and backward reaction rate constants of 6.5 X 10(6) mol-1 X s-1 X l(2) and 3.1 X 10(-4) s-1 X 1, respectively. The kinetic curve also very closely fitted to the experimental data on the basis of the model. The theory for use of the mass action model to optimize the binding-site concentration to give a minimum absolute error for any measured ligand concentration is presented. The use of the model in assay design, standard curve fitting, quality control and problem solving are discussed.

Animals↗
PubMed1990

Establishment of a long-term primary culture of striatal neurons.

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

Animals↗
PubMed1998

Prolongation and optimization of Doppler enhancement with a microbubble US contrast agent by using continuous infusion: preliminary experience.

PURPOSE: To investigate whether continuous infusion of an echo-enhancing contrast agent for up to 15 minutes can provide uniform and prolonged enhancement. MATERIALS AND METHODS: Six volunteers each received one bolus and three infusions of a microbubble contrast agent over 6-15 minutes at (a) a standard rate (mean, 2.08 mL/min), (b) a fast rate at twice the standard rate, and (c) a slow rate at half the standard rate. Spectral Doppler intensitometry of the femoral artery was performed for all infusions. Spectral Doppler ultrasound (US) scans of the femoral artery and color Doppler US scans of the carotid artery were subjectively assessed. RESULTS: All infusions provided an equilibrium plateau of constant prolonged enhancement starting after 1-2 minutes and lasting until the end of the infusion. Enhancement at the plateau was +13 dB (slow rate), +17.1 dB (standard rate), and +18.3 dB (fast rate) compared with baseline. Saturation artifacts with infusions were markedly fewer than those with bolus injections. Dose effectiveness (duration of enhancement that measured at least 7.5 dB per gram of contrast agent) was markedly improved with the infusions, from 0.8 min/g for the bolus to 2.6 min/g for the slow infusion. CONCLUSION: Continuous infusion of the microbubble contrast agent provided prolonged and uniform enhancement of Doppler signals and improved image quality by minimizing saturation artifacts.

Adult↗
PubMed1991

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions.

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.

Aluminum↗
PubMed2004

A theoretical study of the conformational behavior of analogues of alpha-L-rhamnose-1-phosphate.

The conformational behavior of methyl(2-O-methyl-alpha-L-rhamnopyranosyl)phosphate, together with a group of potentially more stable analogues, was investigated through a DFT approach at the B3LYP/6-31G(d) level; the energy of all the optimized structures was recalculated using a continuum solvent model, C-PCM, choosing water as the solvent. The compounds exhibited several, sometimes tenths of populated conformations so that the overall properties of flexibility and mobility were evaluated. The analogue in which the pyranose oxygen atom is replaced by a methylene group emerges as the best candidate as a mimic of the reference 1-phosphate, in spite of the fact that it lacks the anomeric and exo-anomeric effects. The other analogues result poorer mimics because of a conformational equilibrium at the pyranose ring or of an excessive rigidity of the aglycone moiety.

Carbohydrate Conformation↗
PubMed2004

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.

Animals↗
PubMed2004

Probing the pharmacophore for allosteric ligands of muscarinic M2 receptors: SAR and QSAR studies in a series of bisquaternary salts of caracurine V and related ring systems.

Allosteric effects on muscarinic acetylcholine M(2) receptors were examined in a series of bisquaternary salts of the Strychnos alkaloid caracurine V (6) and related iso-caracurine V, tetrahydrocaracurine V, and bisnortoxiferine ring systems. The compounds inhibited dissociation of the orthosteric antagonist [(3)H]N-methylscopolamine (NMS) from porcine cardiac M(2) receptors with EC(0.5,diss) values from 4 to 3270 nM. The majority of compounds hardly changed [(3)H]NMS equilibrium binding, indicating similar binding affinities in free and NMS-occupied M(2) receptors. The most potent agents were found in the caracurine V, iso-caracurine V, and tetrahydrocaracurine V series and carried nonpolar alkyl groups with a maximal chain length of three carbon atoms. 3D QSAR (CoMSIA) analysis explained the wide range of binding affinities by steric and electrostatic properties of the side chains. Furthermore, the findings suggest that the spatial orientation of the "caracurine" aromatic rings compared with the bisnortoxiferine ring skeleton is favorable to optimal allostere-receptor interactions.

Alkaloids↗
PubMed1996

Inhibin binding sites in bovine pituitary membranes.

Membranes derived from bovine pituitary glands free of the neural lobe were used to investigate the presence of binding sites for inhibin, a glycoprotein produced by the ovarian granulosa cells capable of selectively suppressing FSH secretion from the pituitary gland. Optimal concentration of membranes (400 micrograms prot) and 125I-bovine inhibin (2 nM) were incubated in a medium containing 50 mM Tris-HCl pH 7.4, 0.01 M MgCl2 and BSA 0.01% in a final assay volume of 200 microliters at 37 degrees C for different time intervals. Non-specific binding was estimated using unlabelled inhibin in excess. The time course of specific 125I-bovine inhibin (2 nM) binding to bovine pituitary membranes is slow with 50% binding at approximately 20 min of incubation and reaching equilibrium at 90 min of incubation. The kinetic analysis shows an apparent pseudo first order association rate constant (Kob) equivalent to 4 x 10(-2) min-1. Following equilibrium with the tracer, a large excess of unlabelled inhibin (1.27 microM) was able to displace 84% of the specific binding within 120 min of incubation and 50% of the binding at approximately 40 min. The analysis under displacing conditions showed an apparent dissociation rate constant (K2) equals to 1.5 x 10(-2) min-1 and an apparent association rate constant (K1) equals to 1.3 x 10(9) M min-1. Thus, the estimation of the apparent kinetic equilibrium dissociation constant (Kd = K2/K2) of the binding of inhibin to bovine pituitary membranes was 1.2 nM. These results show for the first time the existence of bovine inhibin specific binding sites in bovine pituitary, and also that such a binding can take place in the absence of either gonadal and/or hypothalamic influences. They also contribute to the better understanding of the role of non-steroidal hormones such as inhibin, in the regulation of gonadotrophin secretion.

Animals↗