Biomedical subjects
Publications and source records attributed to M J Alonso.
Based upon the description of the clinico-pathological characteristic of a case with isolated ventricular inversion plus other cardiac malformations, the clinical, diagnostic and embryo-pathological implications of this entity are reviewed.
Ca2+-agonists, Bay K 8644 and CGP 28392, produced concentration-dependent contractions in cat middle cerebral arteries. Only Bay K 8644 induced contractile responses in femoral arteries when they were partly depolarized with 15 mM K+. Nifedipine (0.3 microM) caused a reduction of the contractions elicited by low concentrations of both Ca2+ agonists. In femoral arteries, Bay K 8644 (0.1 microM) increased the contractions elicited by K+ concentrations from 25 to 120 mM, whereas CGP 28392 (0.1 microM) only potentiated the response caused by 25 mM K+. The increase of K+-responses induced by Bay K 8644 was blocked by nifedipine (0.1 microM), whereas that produced by CGP 28392 was not only antagonized but the remaining contractions were less than those obtained in the control situation. In both kinds of arteries preincubated with [3H]noradrenaline, nifedipine (50 microM), CGP 28392 (1 microM and 10 microM) and Bay K 8644 (1 microM) neither modified the spontaneous tritium release nor the stimulated tritium secretion elicited by K+ (25, 50 and 75 mM) or electrical stimulation. These data suggest that CGP 28392 and, mainly, Bay K 8644 facilitate the Ca2+ entry into smooth muscle cells, through voltage-dependent Ca2+ channels (that appear to be preactivated in cerebral arteries), whereas those present in the perivascular adrenergic nerve terminals are unaffected. This fact indicates that the channels existent in both vascular structures seem to have different properties.
A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
The effectiveness of nanoparticle-bound ampicillin was tested in the treatment of experimental Listeria monocytogenes infection in congenitally athymic nude mice. Nanoparticles of polyisohexylcyanoacrylate (PIHCA) 187 +/- 13 nm in diameter were bound to ampicillin at an ampicillin/PIHCA ratio of 0.2:1. The proportion of ampicillin bound was 90% +/- 3%. After adsorption onto nanoparticles, the therapeutic activity of ampicillin increased dramatically over that in the free state. Thus, 2.4 mg of nanoparticle-bound ampicillin (three doses of 0.8 mg each) had a greater therapeutic effect than 48 mg of free ampicillin (three doses of 16 mg each). These results might provide an incentive for further development of intracellular targeting of antibiotics on biodegradable polymeric carriers.
We have partially characterized an intracellular fraction from Phycomyces blakesleeanus which shows proteolytic activity. The apparent thermal inactivation constant (Kd) was 0.12 min-1 at 50 degrees C. This proteolytic fraction was split into two active fractions by ultrafiltration using a membrane with an exclusion size of 30,000. Both fractions were inhibited by phenyl methyl sulphonyl fluoride. The Ki value for the fraction with molecular weight greater than 30,000 was 0.075 mM. The fraction with molecular weight less than 30,000 inactivated the Phycomyces CPS.
Different penicillins (phenylacetyl, 2-hydroxyphenylacetyl, 4-hydroxyphenylacetyl, phenoxyacetyl and 2-thiopheneacetylpenicillin) have been synthesized "in vitro" by direct N-acylation of 6-aminopenicillanic acid (6-APA) with the acyl group of several acyl-CoA derivatives. The enzyme that catalyzes these reactions, acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum, was purified to homogeneity (374-fold) and its amino acid composition is given. This protein accepts as substrates several aliphatic acids and different aromatic acids with the only requirement that an acetyl-CoA moiety must be present in the substrate molecule. Shortening or lengthening of the acyl moiety prevents the 6-APA-N-acylation reaction. The presence of an amino group in the alpha-position of the acetyl group does not allow this molecule to be used as substrate. However, different substitutions in the phenyl group (hydroxylation of the carbons 2 and 4) or its replacement by another aromatic ring (thiophene) were accepted with varying reactions rates in the acylation reaction when a 176-fold purified acyltransferase was employed. The homogeneity pure enzyme accepts as substrate thiophene acetyl-CoA but it did not 2-hydroxyphenyl and 4-hydroxyphenylacetyl-CoA. The presence of an oxygen atom between the aromatic and the acetyl moieties did not affect the catalysis.
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Rhinosporidiosis is reviewed, and the first autochthonous case in Spain is presented (site: in the nasal cavity of a 19-year-old male from a rural background). Diagnosis was established morphologically after eliminating the possibilities of Cryptococcus neoformans, Coccidioides immitis, and Chrysosporium crescens. Clinico-pathological features are described. Preparations were stained with hematoxylin-eosin, PAS, and methenamine silver, and studied for fluorescence. Certain aspects of the epidemiology and diagnosis are commented upon.
The incorporation of L-[U-14C]valine into delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a direct biosynthetic precursor of penicillins and cephalosporins, was studied. When DL-methionine was added to Acremonium chrysogenum culture broths, no labeled ACV was found, while a large amount of radioactive ACV was detected when methionine was not present. DL-Norleucine, a nonsulfur analog of methionine, also inhibited the synthesis of radioactive ACV to some degree. This effect was due to the inhibition of valine transport by methionine and norleucine.
A controlled study of the bioavailability of paracetamol in solid dispersion with PEG 6000, 10000 and 20000 has been made. The total amount of paracetamol excreted in urine increased with molecular weight of the PEG, but the rate of absorption of the drug was unaffected.
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Currently, in certain clinical situations there is an increasing trend towards using dosage regimens involving aminoglycoside antibiotics based on the administration of a single dose of the drug per day instead of administering the same amount in two or three administrations. The aim of the present study was to discover the pharmacokinetic profile and the nephrotoxic potential of this new form of administration in experimental animals receiving gentamicin. The study was conducted on two groups of rabbits, one of which received a single dose of the drug at 7 mg/kg i.v. and the other 7 mg/kg administered every 12 hours, allometrically equivalent to gentamicin dosing at 5 mg/kg every 24 hours to human subjects. The number of doses administered was 20. From the pharmacokinetic point of view, the results point to the existence of a significant degree of accumulation of the antibiotic in renal cortex as a result of the dosage regimen, no important modifications occurring in the pharmacokinetic parameters of gentamicin calculated from its plasma kinetics. This shows that the two compartment model employed predicts drug levels in accessible tissues but not in deep ones where gentamicin is accumulated for long periods of time. From the toxicological point of view, the treatment caused appreciable damage of the renal tubules during the first phases of the treatment which was not detectable from the serum creatinine levels or the kinetic behaviour of the aminoglycoside.
The high aqueous solubility of chitosan restricts the utility of chitosan microspheres for gastric drug delivery. This paper describes the preparation of reacetylated chitosan microspheres with suitable properties for the controlled release of active anti-microbial agents, such as amoxycillin and metronidazole, in the gastric cavity. Two different microencapsulation approaches were developed and optimized in order to encapsulate hydrophilic (amoxycillin) and hydrophobic (metronidazole) compounds efficiently. The reacetylated chitosan microspheres exhibited a controlled water swelling capacity and gelified at acidic pH, resulting in prolonged release of the encapsulated antibiotics. The reacetylation time was found to be a key factor that affects not only drug release, but also encapsulation efficiency and anti-microbial activity of the encapsulated compound. The last two parameters were also dependent on drug solubility in the reacetylating agent. Using short reacetylation time periods, it was possible to efficiently control the release of both hydrophilic and lipophilic antibiotics while maintaining their activity against different bacteria. Consequently, reacetylated chitosan microspheres are promising vehicles for the controlled delivery of anti-microbial agents to the gastric cavity and, hence, for the eradication of Helicobacter pylori, a pathogen strongly associated with gastric ulcers and possibly gastric carcinoma.
The possible contribution of nitric oxide (NO) and other endothelial factors to the vasodilatation induced by acetylcholine (ACh) was analyzed in cat femoral arteries. For this purpose, the modifications by different treatments of ACh-induced relaxations were compared with those of exogenous NO-induced relaxations. Although the ACh-induced endothelium-dependent relaxation was not affected by the cyclooxygenase inhibitor indomethacin, it was partially inhibited by the lipoxygenase blocker 5,8,11,14-eicosatetraenoic acid, and by the NO inactivators phenidone, hydroquinone and oxyhemoglobin (OxHb). Additionally, this type of relaxation was reduced by the NO-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) and abolished by NG-nitro-L-arginine methyl ester; these later effects were selectively antagonized by L-arginine. Exogenous NO-induced vasodilatations were abolished by OxHb and unmodified by L-NMMA. Blocking sodium pump activity decreased vasodilatations to ACh, NO, sodium nitroprusside and 8-bromoguanosine 3',5'-cyclic monophosphate. 4-Aminopyridine, a K+ channel antagonist, reduced the relaxation induced by ACh but not that induced by exogenous NO. These results suggest: (1) ACh-induced relaxation in these vessels is mainly due to endothelial cell NO release; (2) NO does not act on 4-AP-sensitive K+ channels, although these channels may be involved in NO release from the endothelium, and (3) one of the relaxant mechanisms of NO could be the activation of the vascular sodium pump.
We have recently reported preliminary data showing the efficacy of chitosan nanocapsules as carriers for oral peptide delivery. In the present work, our aim was to investigate the influence of some chitosan properties, such as the molecular weight and type of salt, on the interaction of these nanocapsules with the Caco-2 cells and also on their in vivo effectiveness. Chitosan nanocapsules were prepared by the solvent displacement technique using high (450 kDa) and medium (160 kDa) molecular weight chitosan glutamate as well as high molecular weight chitosan hydrochloride (270 kDa). The results indicated that the size of the nanocapsules was dependent on the chitosan molecular weight, whereas the zeta potential and the association efficiency of salmon calcitonin were not affected by the chitosan properties. Upon incubation with the Caco-2 cells, chitosan nanocapsules exhibited a dose-dependent cellular viability, which was hardly affected by, either the chitosan molecular weight or, the type of salt. In addition, it was observed that the transepithelial electrical resistance of the Caco-2 monolayer was not significantly modified upon their exposure to chitosan nanocapsules. The results of the in vivo studies, following oral administration to rats, indicated that chitosan nanocapsules were able to reduce significantly the serum calcium levels, and to prolong this reduction for at least 24 hours, irrespective of the type of chitosan salt and molecular weight of chitosan. Consequently, the performance of chitosan nanocapsules as oral carriers for salmon calcitonin was not affected by the characteristics of chitosan.
The aim of the present work was to develop a new nanoparticle carrier, adapted for the oral administration of proteins and their delivery to the immune system. Chitosan and phosphorylated glucomannan were chosen as major constituents of the nanoparticles. Chitosan nanoparticles were formed by ionic gelation and then coated with glucomannan. Two different protocols were adopted for the formation of the glucomannan coating: protocol I, in which chitosan nanoparticles were isolated before their coating; protocol II, in which chitosan nanoparticles were not isolated, but coated with glucomannan in the presence of free chitosan. The results showed that, under the selected formulation conditions, the sizes of the nanoparticles ranged between 170 and 300 nm and their zeta potential values were inverted from positive to negative by the glucomannan coating. The nanoparticles prepared by the two protocols could be freeze-dried, in the presence or absence of cryoprotective agents, preserving their original characteristics. The results of the stability study evidenced the positive role of the glucomannan coating in preventing the aggregation of the nanoparticles in buffered media. Finally, the association of the inmunomodulatory protein complex P1 to the chitosan-glucomannan nanoparticles was investigated. The results showed that the association was not dependent on the chitosan: sodium tripoliphosphate ratio, but it was significantly affected by the presence of sodium phosphate in the protein structure.
A rotatable central composite design was applied to the formulation of poly(butyl 2-cyanoacrylate) nanoparticles with the aim of modifying some physico-chemical properties of these carriers in a controlled way. The joint effect of stabilizer concentration and monomer concentration on the mean particle size, on the electrophoretic mobility and on the polymer molecular weight has been investigated. The results demonstrate that the particle size is only dependent on the stabilizer concentration, nevertheless the electrophoretic mobility and the molecular weight distribution were affected simultaneously by the monomer and the stabilizer concentrations. Moreover, using the response surface diagrams it is possible to deduce the experimental conditions to design particles with the desired properties. Finally, the degradation rates for two different molecular weight formulations were compared, showing the relevance of this property.
Cefuroxime axetil (CA) was encapsulated in pH-sensitive acrylic microspheres in order to formulate a suspension dosage form. Using this microencapsulated form it was expected to prevent leaching of the drug from the microspheres into the suspension medium and to assure the release of the drug in the first part of the intestine, thus avoiding changes to its bioavailability. For this purpose, CA was microencapsulated within several types of acrylic polymers by the solvent evaporation and the solvent extraction techniques. The acrylic polymers selected were: Eudragit E (positively charged and soluble at pH 5), Eudragit L-55 (negatively charged and soluble at pH > 5.5) and Eudragit RL (neutral, insoluble, but readily permeable). The influence of the polymer electrical charge on the stability and in vitro release of CA was investigated. Though Eudragit E microspheres presented good morphological characteristics and dissolution behaviour, the analysis of the stability of CA in the presence of Eudragit E by HPLC, indicated a negative interaction between both compounds. However, formulations made of Eudragit L-55 and RL in the ratios 100:0 and 90:10 were adequate in terms of the stability of the encapsulated CA. The dissolution studies showed a critical pH between 5.2 and 6.0, which allowed the complete release of CA in a short period. Furthermore, these polymer microspheres were shown to be efficient in masking the taste of CA.