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Biomedical subjects

L Saso

Publications and source records attributed to L Saso.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of lonidamine in rat serum and testis by high performance liquid chromatography.

A HPLC method for the determination of lonidamine in serum and testis, suitable for pharmacological studies in the rat and other mammals, has been developed. Briefly, 0.5 mL of serum or about 0.2 g of testicular tissue were extracted with ethyl acetate and evaporated to dryness under nitrogen. The residue was redissolved in methanol and an aliquot was injected onto a C18 column eluted with a mobile phase consisting of acetonitrile:water (51:49, v/v), containing 0.1% trifluoroacetic acid. The eluate was monitored at 230 nm with a sensitivity of 0.05 AUFS. By this method, the pharmacokinetics and the serum and testicular levels of the drug up to 120 h after the administration of one single dose (100 mg/kg body weight) of lonidamine to Sprague-Dawley rats have been studied. Results were highly variable, as previously reported, but a very good linear correlation was found between the serum and the testicular levels, suggesting that, in the rat, and possibly in the human, testicular levels could be estimated based on the serum concentrations.

Animals↗
PubMed2001

Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): pharmacological implications.

The activity of nonsteroidal antiinflammatory drugs (NSAIDs) in rheumatoid arthritis is not only due to the inhibition of the production of prostaglandins, which can even have beneficial immunosuppressive effects in chronic inflammatory processes. Since we speculated that these drugs could also act by protecting endogenous proteins against denaturation, we evaluated their effect on heat-induced denaturation human serum albumin (HSA) in comparison with several fatty acids which are known to be potent stabilizers of this protein. By the Mizushimas assay and a recently developed HPLC assay, we observed that NSAIDs were slightly less active [EC50 to approximately 10(-5)-10(-4) M] than FA and that the HPLC method was less sensitive but more selective than the turbidimetric assay, i.e. it was capable of distinguishing true antiaggregant agents like FA and NSAIDs from substances capable of inhibiting the precipitation of denatured protein aggregates. In conclusion, this survey could be useful for the development of more effective agents in protein condensation diseases like rheumatic disorders, cataract and Alzheimers disease.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Inhibition of calcium oxalate precipitation by bile salts.

BACKGROUND: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former. METHODS: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method. RESULTS: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate. CONCLUSION: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.

Bile Acids and Salts↗
PubMed2001

Antidenaturant drugs for cataract and other condensation diseases.

'Condensation diseases' are heterogeneous pathological conditions in which the primary pathogenetic step is the loss of solubility of specific substances, resulting in the formation of a condensed phase. Typical examples are cataract, nephrolithiasis, gallstone disease and certain rheumatic conditions in which protein denaturation, aggregation and precipitation may occur. Since the condensing molecules are often proteins, antidenaturant agents should be considered rational drugs for the treatment of these diseases. Surprisingly, however, only a few molecules with these properties are currently available for therapeutic use, including bendazac for cataract.

Cataract↗
PubMed2001

Two new male contraceptives exert their effects by depleting germ cells prematurely from the testis.

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3-7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4-14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

Animals↗
PubMed2000

In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography.

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimer's disease.

Antioxidants↗
PubMed2000

Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography.

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.

Animals↗
PubMed2000

Antimicrobial investigation of semipurified fractions of Ginkgo biloba leaves.

A total methanolic extract of Ginkgo biloba leaves was fractionated by solvent partition using ethyl acetate (fraction A), n-butanol (fraction B) and water (fraction C). The antimicrobial activity of the three fractions was evaluated using a number of Gram-positive and -negative bacteria and yeasts. The apolar fraction A appeared to be the most interesting because of its activity against several microorganisms; this fraction was further separated by high performance liquid chromatography, and shown to contain substances with strong inhibitory activity against Enterococcus faecalis 31, different from the major known chemical components of G. biloba leaves.

Anti-Bacterial Agents↗
PubMed2000

Stimulation of guinea pig isolated atria by aflatoxins.

Acute effects of aflatoxins (AF), and in particular cardiac actions, have not been examined as much as chronic toxicity. Thus, in the present study we evaluated the effects of specific AF on isolated guinea pig atria. Isoprenaline (ISO, 4x10(-9)), AFB(1) (3x10(-6) and 6x10(-5) M) and AFG(1) (3x10(-6) and 6x10(-6) M) contracted the isolated guinea pig atria, leaving the preparation hyperresponsive to ISO. These properties of AF are of interest as they could be responsible of certain cardiotoxic effects described in the literature.

Aflatoxins↗
PubMed2000

Relaxant effects of Hydrastis canadensis L. and its major alkaloids on guinea pig isolated trachea.

Hydrastis or goldenseal, one of the most popular medicinal herbs in the U.S.A., is used in mild pathological conditions like cold and flu, based on the pharmacological properties of its active components, berberine (anticholinergic, antisecretory, and antimicrobial) and beta-hydrastine (astringent). We previously reported the relaxant effect of a total ethanolic extract of hydrastis on carbachol precontracted isolated guinea pig trachea, and with the present study, using the same experimental model, we aimed at evaluating the contribution of its major alkaloids, berberine, beta-hydrastine, canadine and canadaline to the total effect. Furthermore, using specific pharmacological tools, like timolol and xanthine amine congener, we attempted to elucidate its mechanism of action. The EC50 of berberine, beta-hydrastine, canadine and canadaline, were 34.2+/-0.6, 72.8+/-0.6, 11.9+/-1.2 and 2.4+/-0.8 microg/ml, respectively. Timolol effectively antagonized the effect of canadine (EC50 = 19.7+/-3.0 microg/ml) and canadaline (EC50 = 17.1+/-1.2 microg/ml) but not that of berberine and beta-hydrastine, while xanthine amine congener antagonized the effect of beta-hydrastine (EC50 = 149.9+/-35.3 microg/ml) and canadaline (EC50 = 26.1+/-3.0 microg/ml) but not that of berberine and canadine. Besides, the hydrastis extract, at concentrations between 0.01 and 0.1 microg/ml, potentiated the relaxant effect of isoprenaline on carbachol-precontracted isolated guinea pig trachea. These data, which are insufficient to draw definite mechanistic conclusions, indicate that the aforementioned alkaloids may act by interacting with adrenergic and adenosinic receptors.

Alkaloids↗
PubMed2000

Aflatoxins inhibit prolactin secretion by rat pituitary cells in culture.

Acute effects of aflatoxins (AF), and in particular hormonal actions, have not been examined as much as chronic toxicity. Thus, we studied the effects of specific AF on prolactin (PRL) secretion by rat pituitary cells in culture. AFB1 and AFQ1 (1 x 10(-4) M) reduced the stimulating effect of dimethyl sulfoxide on PRL secretion by cultured rat pituitary cells. The mechanism responsible for this action is still unknown, but it did not seem to be a non specific toxic effect, because AFB1, at the same concentration, did not significantly alter cell viability, as indicated by the Trypan blue dye-exclusion test.

Aflatoxin B1↗
PubMed2000

Relaxant effects of aflatoxins on isolated guinea pig trachea.

Dyspnea is one of the symptoms of acute aflatoxicosis. Contrary to expectations, we observed that naturally occurring aflatoxins (AF) AFB(1), AFB(2), AFG(1), and AFG(2) and their major metabolites AFM(1), AFM(2), AFP(1), AFQ(1), and AFG(2a) relaxed carbachol (C) precontracted guinea pig trachea to different degrees. The efficacies but not the potencies of AFB(1), AFB(2), AFG(1), and AFG(2) were similar to that of the beta-agonist, isoprenaline, whose activity was potentiated by the AF. Their mechanism of action is not clearly understood but several mechanistic indications were obtained with AFB(1): 1) its effect was not influenced by the beta-blocker, timolol, indicating that a direct interaction with beta(2)-adrenergic receptors was not involved. 2) AFB(1) potentiated PGE(1) and PGE(2), two relaxant prostaglandins, and its activity was reduced by indomethacin. 3) The cAMP level in the guinea pig trachea relaxed by AFB(1) increased, possibly due to inhibition of phosphodiesterase; direct interaction with PG receptors; and/or interaction with A(2) adenosinic receptors, suggested by the inhibitory activity of XAC, a specific antagonist. 4) Finally, since tetrodotoxin reduced the relaxant activity of AFB(1), it is speculated that this mycotoxin could stimulate inhibitory nonadrenergic, noncholinergic nerves (i-NANC). In conclusion, the symptoms of acute aflatoxicosis do not seem to be due to a direct activity on the tracheal muscle, but rather, to the well-known pro-inflammatory activity of the aflatoxins, which are capable of releasing arachidonic acid from cell membranes.

Aflatoxins↗
PubMed2000

Inhibition of heat-induced aggregation of beta- and gamma-crystallin by alpha-crystallin evaluated by gel permeation HPLC.

The capability of alpha-crystallin (alpha-C), a known molecular chaperon, of protecting beta-C and gamma-C against heat-induced aggregation was studied by gel permeation high performance liquid chromatography. The activity was calculated using a formula based on the changes in the areas under the chromatographic peaks of these proteins, which appeared well separated. When heat-induced aggregation was studied in the range 22-90 degrees C, beta-C appeared more stable than gamma-C. The activity of alpha-C in stabilizing gamma-C but not beta-C was already relevant at 60 degrees C, but the maximum activity was higher (about 35%) for beta-C than for gamma-C. This method could be useful for studying the effect of drugs with potential anti-cataract activity on heat-induced aggregation of individual lens proteins.

Cataract↗
PubMed2000

Changes of acute-phase proteins in streptozotocin-induced diabetic rats.

Quantitative and qualitative changes of serum proteins, apart from glycation, have not been sufficiently studied in streptozotocin-induced diabetic rats (D), the most common experimental model for diabetes. Thus, we decided to analyze the serum of diabetic rats by concanavalin A-blotting in comparison with rats with acute inflammation induced by fermented yeast (Y), in which characteristic alterations of serum proteins have been described. Two months after the streptozotocin treatment, the blood glucose levels were highly elevated (456+/-24 vs. 124+/-10 mg/dl, p<0.001, n=12), the body weight was significantly lower than normal (279+/-10 vs. 392+/-6 g, p<0.001, n=12), and serum proteins appeared to be highly glycated (p<0.001) when analyzed by the fructosamine assay, without any significant change in the total serum protein concentration. Analysis by concanavalin A-blotting, revealed a significant decrease of alpha1-inhibitor-3 (alpha1-I3, p<0.05) and an increase of the beta chain of haptoglobin (beta-Hp, p<0.05) in both D and Y rats (n=3) compared with control animals. However, acute inflammation caused a marked rise of two prominent acute phase proteins, alpha2-macroglobulin and hemopexin, which did not change appreciably in diabetic rats. Further work will be necessary to evaluate the physiopathological significance of these phenomena which could result from changes of both concentration and glycosylation of the aforementioned proteins.

Acute-Phase Proteins↗
PubMed2000

[Ethical issues of human experimentation with special reference to informed consent].

Human experimentation in order to develop new medical therapies creates very complex ethical problems: when is it possible to test a new therapy on a human subject? Is it always necessary his/her consent? Which information should be given to the subject before requesting his/her consent? How to behave in the case of minors, psychiatric patients and other subjects not perfectly free or able to understand the information provided? Is it right to subject a person to an experimentation from which he/she will not get any direct advantage? Which results can be published? In other words, which are the ethical limits of human experimentation? These are difficult questions, to which the authors tried to answer referring to some ethically significant human experimentations, such as those performed by Lind and Jenner in the XVIII century, and those carried out by the nazi doctors, from whose trial derived the Nuremberg Code, which introduced for the first time, at an international level, the principle of the informed consent. Some of the limits of this document, including the impossibility of doing research on subjects not able to give their informed consent, such as minors and psychiatric patients, were overcome by the Declaration of Helsinki, whose current version resulted from several subsequent revisions. According to this document, a major role is played by the Institutional Review Boards or Institutional Ethical Committees that have the heavy responsibility of evaluating the ethical connotations of human experimentations.

Ethics, Medical↗
PubMed1999

Changes in concanavalin A-reactive proteins in neurological disorders.

Changes of glycosylation of cerebrospinal fluid proteins such as alpha2-macroglobulin, and prostaglandin D synthase were studied by lectin blotting, using concanavalinA, in multiple sclerosis (n = 42) and neuropathies (n = 20) in comparison to neurological controls (n = 22). The concanavalinA-reactivity of alpha2-macroglobulin, which was increased in the neuropathies but not in multiple sclerosis compared to controls, correlated with the total concanavalinA-reactivity in controls and neuropathies but not in multiple sclerosis, indicating that the protein could be abnormally glycosylated in the latter disease. Although the concentration and the concanavalinA-reactivity of prostaglandin D synthase were not significantly different in the three groups, the two parameters correlated only in neuropathies but not in controls or multiple sclerosis, probably due to the high heterogeneity of the protein. These changes deserve to be studied in further detail in view of their potential clinical applications.

Blood Proteins↗
PubMed1999

Stabilization of rat serum proteins following oral administration of fish oil.

The mechanism of action of fish oil (FO), currently used in different chronic inflammatory conditions such as rheumatoid arthritis (RA), is not completely understood, although it is thought that it could alter the metabolism of endogenous autacoids. In addition, we hypothesized that the known capability of fatty acids (FA) of stabilizing serum albumin and perhaps other proteins, may be of pharmacological relevance considering that it is shared by other anti-rheumatic agents (e.g. nonsteroidal antiinflammatory drugs). Thus, we studied the effect of oral administration of FO and corn oil (CO), a vegetable oil with a different composition, on the stability of rat serum proteins, evaluated by a classical in vitro method based on heat-induced protein denaturation. FO, and, to a lower extent, CO inhibited heat-induced denaturation of rat serum (RS): based on the inhibitory activity (EC50) of the major fatty acids against heat-induced denaturation of RS in vitro, it was possible to speculate that in vivo effects of palmitic acid (C16:0) and eicosapentaenoic acid (EPA, C20:5, n-3) may be more relevant than that of linolenic acid (C18:2). To better investigate this phenomenon, we extracted albumin from the serum of animals treated or not with FO with a one-step affinity chromatography technique, obtaining high purity rat serum albumin preparations (RSA-CTRL and RSA-FO), as judged by SDS-PAGE with Coomassie blue staining. When these RSA preparations were heated at 70 degrees C for 30 min, it was noted that RSA-FO was much more stable than RSA-CTRL, presumably due to higher number of long chain fatty acids (FA) such as palmitic acid or EPA. In conclusion, we provided evidences that oral administration of FO in the rat stabilizes serum albumin, due to an increase in the number of protein bound long chain fatty acids (e.g. palmitic acid and EPA). We speculate that the stabilization of serum albumin and perhaps other proteins could prevent changes of antigenicity due to protein denaturation and glycosylation, which may trigger pathological autoimmune responses, suggesting that this action may be involved in the mode of action of FO in RA and other chronic inflammatory diseases.

Administration, Oral↗