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Despite the risk of kidney damage, lithotripsy is the usual way of treating calcium oxalate (CaOx) stones, the most common type of nephrolithiasis, because no effective chemolytic agents are available. However, the search of new calcium chelators, less toxic than the current ones, continues, and some of them could be tested in experimental models of nephrolithiasis, after their ability of dissolving CaOx crystals is verified. In this connection, we developed a simple assay that requires only inexpensive equipment available in most laboratories for the screening of substances potentially capable of dissolving CaOx crystals. In particular, we decided to investigate whether substances previously shown to inhibit CaOx precipitation were also capable of dissolving this salt. Briefly, CaOx tablets of highly reproducible weight (4.55 ± 0.07 mg) were prepared by spinning, at high speed (16,000 g), microcentrifuge tubes in which 500 µl aliquots of 0.1 M sodium oxalate and 0.1 M calcium chloride at pH 6 were added. When these tablets were incubated overnight with solutions at different concentrations of EDTA, sodium citrate, manganese chloride, sodium sulfate, sodium chloride, malic acid, succinic acid and gluconic acid, a significant dissolving activity was observed for EDTA (∼25% at 0.25 M), sodium citrate (∼30% at 1 M) and manganese chloride (∼20% at 0.5 M). A good linear correlation (r[sup 2] = 0.84, p < 0.05) was found between the affinity for calcium and the activity of EDTA, sodium citrate, sodium sulfate, malic acid, succinic acid and gluconic acid, indicating that these compounds act mainly by chelating the calcium ion. Instead, manganese was supposed to act by interacting with the oxalate ion. [ABSTRACT FROM AUTHOR]

Introduction: Cardiovascular diseases (CVD) are a leading cause of death worldwide, and several modifiable and unmodifiable risk factors contribute to this burden of disability and mortality. Thus, effective cardiovascular prevention relies on appropriate strategies to control risk factors within the frame of unmodifiable traits., Methods: We conducted a secondary analysis of treated hypertensive adults aged ≥ 50 years enrolled in Save Your Heart. CVD risk and hypertension control rates based on the 2021 updated European Society of Cardiology guidelines were evaluated. Comparisons with previous standards in terms of risk stratification and hypertension control rates were performed., Results: Among the 512 patients evaluated, with the application of the new parameters for fatal and non-fatal cardiovascular risk assessment, the proportion of individuals at high or very high risk rises from 48.7 to 77.1% of cases. A trend towards lower hypertension control rates was observed based on 2021 European guidelines compared with the 2018 edition (likelihood estimate for difference: 1.76%, 95% CI - 4.1 to 7.6%, p = 0.589)., Conclusions: In this secondary analysis on the Save Your Heart study, the application of the new parameters reported in the European Guidelines for Cardiovascular Prevention 2021 showed a hypertensive population with a very high probability of encountering a fatal or non-fatal cardiovascular event due to failure to control risk factors. For this reason, a better management of risk factors must be the main goal for the patient and all the involved stakeholders., (© 2023. The Author(s).)

The need for quick tools to sketch an early but accurate cognitive profile of patients who suffered brain damage or head trauma is of primary importance. Nonetheless, in the Italian context, the most-diffused screening tools are still those originally devised to diagnose dementia. The present pilot study then aimed at investigating the potential and feasibility of a novel screening battery, the Cognitive Assessment for Stroke Patients (CASP), in a sample of Italian patients by comparing it to Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) tests. A total of 29 neurology patients took part in the study. Participants underwent a screening procedure including the administration of MMSE, MoCA, and CASP. Data analysis suggested that the scores of the Italian version of the CASP are relatively less affected by the presence of language difficulties-common sequelae of stroke and head traumas-with respect to MMSE and MoCA ones. Furthermore, CASP scores proved to be highly correlated with both MMSE and MoCA scores, showing good clinical potential. Finally, the outcomes of administered tests proved not to be influenced by etiology or gender, and CASP scores showed a diminishing trend related to patients' age and a positive association with patients' education.

Individuals with multiple sclerosis (MS) often face important health-related and financial decisions that involve trade-offs between short-term and long-term benefits, yet decision making is rarely studied in MS patients. The temporal discounting paradigm is a useful tool for investigating such time-dependent choices in humans. Here, we investigated whether patients with relapsing-remitting MS differed from healthy controls when making choices between hypothetical monetary rewards available at different points in time. Participants were tested in two conditions: in one, the choice was between a smaller amount of money available immediately and a larger amount of money available at a later date; in the other, a fixed delay of 60 days was added to both options. We found that, compared with healthy controls, MS patients favored less the sooner reward in the condition involving an immediate reward, whereas no difference between MS patients and the control group emerged in the condition involving only delayed rewards. Moreover, the decreased immediacy bias was corroborated by lower scores at scale that assesses responsiveness to rewards in MS patients. Taken together, these findings indicate reduced sensitivity to immediate reward and a consequent stronger willingness to defer gratification in MS individuals. (PsycINFO Database Record, ((c) 2017 APA, all rights reserved).)

Introduction: The present study explored the role of resting state and personality component (BIS/BAS measure) on prefrontal cortical responsiveness to emotional cues. Indeed, we supposed that lateralized resting activity (right vs. left) and approach (BAS) versus avoidance (BIS) attitude may explain the successive emotional processing within the prefrontal cortex (PFC) based on the stimulus valence (positive and negative emotional cues)., Methods: Hemodynamic (functional near-infrared spectroscopy, fNIRS) and electroencephalographic (EEG) measures were considered. The resting and experimental brain activity were registered when subjects ( N  = 21) viewed emotional positive versus negative stimuli (International Affective Picture System, IAPS). LIR eeg and LIR nirs (lateralized Index Response) during resting state, and LI eeg and LI nirs during emotional processing were acquired., Results: A set of regression analyses was applied to the multiple measures. The predictive effect of resting activity and approach/avoidance dichotomy were elucidated. Indeed, more left/right resting activity (for both LIR eeg and LIR nirs ) predicted the successive more brain left/right response (LI eeg and LI nirs ) to emotional cues. Second, significant effects were revealed as a function of valence (increased right response to negative stimuli; increased left response to positive stimuli) during emotion processing. Third, higher BAS values explained an increased left cortical activity in resting state and in experimental condition for positive cues. In contrast, higher BIS values marked an increased right activity in resting state and in experimental condition in response to negative cues., Conclusion: The significance of trait component for both resting and emotional cue processing was discussed at light of the present results.

This study explored the effect of lateralized left-right resting brain activity on prefrontal cortical responsiveness to emotional cues and on the explicit appraisal (stimulus evaluation) of emotions based on their valence. Indeed subjective responses to different emotional stimuli should be predicted by brain resting activity and should be lateralized and valence-related (positive vs negative valence). A hemodynamic measure was considered (functional near-infrared spectroscopy). Indeed hemodynamic resting activity and brain response to emotional cues were registered when subjects (N = 19) viewed emotional positive vs negative stimuli (IAPS). Lateralized index response during resting state, LI (lateralized index) during emotional processing and self-assessment manikin rating were considered. Regression analysis showed the significant predictive effect of resting activity (more left or right lateralized) on both brain response and appraisal of emotional cues based on stimuli valence. Moreover, significant effects were found as a function of valence (more right response to negative stimuli; more left response to positive stimuli) during emotion processing. Therefore, resting state may be considered a predictive marker of the successive cortical responsiveness to emotions. The significance of resting condition for emotional behavior was discussed., (© The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.)

Due to its fast temporal evolution and its representation and integration among complex and widespread neural networks, the emotion perception process should preferably be examined by means of multimethodological approach. Indeed the indubitable vantage of acquiring both the autonomic (arousal-related) and the central (cortical-related) activities stands in the possibility to better elucidate the reciprocal interplay of the two compartments. In the present study EEG (frequency band analysis), systemic SCR and heart rate (HR) were all recorded simultaneously with hemodynamic (NIRS, Near-Infrared Spectroscopy) measurements as potential biological markers of emotions, related to both central and peripheral systems. These multiple measures were then related to the self-report correlates, that is the subjective appraisal in term of valence (positive vs. negative) and arousal (high vs. low) by using SAM rating. Twenty subjects were submitted to emotional cues processing (IAPS) when fNIRS, frequency bands (alpha, beta, delta, theta), SCR and HR were recorded. As shown by O2Hb increasing within the right hemisphere, the contribution of prefrontal cortex was elucidated, by pointing out a relevant lateralization effect (more right-PFC activity) induced by the specific valence (negative) of the emotional patterns. Secondly, EEG activity (mainly low-frequency theta and delta bands) was intrinsically associated with the cortical hemodynamic responsiveness to the negative emotional patterns, within the right side. Finally SCR increased mainly in response to negative patterns, and the autonomic behavior was related to explicit (SAM) and cortical (NIRS; EEG) activity. The intrinsic relationships between these three different levels are discussed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A new assay for the screening of hypochlorite/hypochlorous acid (XOCl) scavengers, based on the reversed-phase high performance liquid chromatographic analysis of human serum albumin (HSA, 0.2% in 100 mM sodium phosphate, pH 7), before and after oxidation by XOCl (1.6 mM), was developed. XOCl induced a significant decrease of the area under the chromatographic peak of HSA at 280 nm due to the oxidation of the aromatic amino acids tryptophan and tyrosine, as suggested by the literature and by the chromatographic analyses and the electrochemical study performed here. The assay was validated by testing known XOCl scavengers such as ascorbic acid, cysteine, glutathione, S-methylglutathione and alpha-lipoic acid and other antioxidants such as carnosine and chlorogenic acid, which inhibited the oxidation of HSA. Quantitative activities were calculated using an original formula based on the changes of the area of the albumin peak. Electrochemical data collected here in a homogeneous medium showed that the anodic potentials of the antioxidants tested are less positive (ascorbic acid, chlorogenic acid and cysteine) or similar (alpha-lipoic acid) compared with those of the aromatic residues (tryptophan and tyrosine) of HSA oxidized by XOCl. However, as expected, carnosine, glutathione and S-methylglutathione were inactive at a glassy-carbon, gold or platinum electrode., (Copyright 2002 John Wiley & Sons, Ltd.)

Neither quercetin (Q), nor 3-O-acylquercetines, up to 100 microg/mL, had any significant activity on selected gram-positive strains (Staphylococcus aureus, Bacillus subtilis, Listeria ivanovi, Listeria monocytogenes, Listeria serligeri), gram-negative strains (Escherichia coli, Shigella flexneri, Shigella sonnei, Salmonella enteritidis, Salmonella tiphymurium) and yeasts (Candida albicans and Candida glabrata). In addition, we confirmed the known anti-HIV activity of Q (80% inhibition at 40 microM), which might depend on the free hydroxyl in the C-3 position, as suggested by the lack of activity of the 3-O-acylquercetines. Finally, we described an interesting inhibitory activity on Candida rugosa lipase by Q (IC(16)=10(-4) M) and its esters (3-O-acylquercetines) which, in vivo, could play an important role against lipase producing microorganisms. In particular, 3-O-acyl-quercetines, being more active (IC(16)=10(-4)-10(-6) M) and more lipophilic, could be more effective than Q when applied to the skin or mucosae, and deserve to be studied further.

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.

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.

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.

In a recent study (Leone et al., 2000) we reported that lonidamine (LND), an antispermatogenic drug, affected the concentration of selected testicular and epididymal proteins in the rat. Thus, the effect of LND on alpha2-macroglobulin (alpha2-M) and on other two acute phase proteins (APP), hemopexin (HPX) and alpha1-antitrypsin (alpha1-AT) was examined here. LND was administered orally at the dose of 100 mg/kg, the animals were killed after 24 and 48 hr and the samples were analyzed by immunoblotting. The drug did not induce any significant change of alpha2-M in the serum or testis and of HPX and alpha1-AT in the serum, testis or epididymis. Thus, the antispermatogenic action of LND was not accompanied by a significant change of these inflammatory markers, even if it did cause a decrease of alpha1-inhibitor-3, a negative APP, as previously reported (Leone et al., 2000).

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.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

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.

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.

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.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.

Changes in serum and cerebrospinal fluid (CSF) proteins following generalized acute inflammation induced by fermented yeast in the rat was examined by concanavalin A-blotting, immunoblotting, and radioimmunoassay. Using alpha2-macroglobulin (alpha2-M) and hemopexin (HPX) as marker proteins, the concentration alpha2-M was found to increase in serum and CSF by 150- and 5-fold, respectively, whereas the concentration of HPX increased by about 4-fold in both fluids following yeast-induced inflammation. The lesser increase in alpha2-M in the CSF versus the systemic circulation is not likely to be the result of changes in the permeability of the blood--brain barrier, since no change in the total protein content of CSF was detected in inflamed rats when compared to control animals. These results, however, illustrate the regulation of the same protein, such as alpha2-M, in two separate organs within the same animal can be drastically different. These results also suggest a possible protective role of alpha2-M in the brain during acute inflammation. Moreover, these observations are consistent with the previous observation that there is a differential response in the level of alpha2-M between the testis and the systemic circulation during inflammation.

Objectives: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum alpha2-macroglobulin (alpha2-M) from patients with systemic lupus erythematosus (SLE)., Design and Methods: Serum alpha2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using alpha2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n = 14), SLE (n = 34), rheumatoid arthritis (n = 15), Sjögren's syndrome (n = 11), mixed connective tissue diseases (n = 12), and liver diseases (n = 11) were analyzed., Results: The affinity of the mab for alpha2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA., Conclusions: The ELISA was capable of recognizing changes of glycosylation of alpha2-M in SLE and may be useful for its differential diagnosis.

Although it is well established that ocular mucins and other proteins are essential for tear film stability, whether certain drugs, like non steroidal antiinflammatory drugs (NSAIDs), could cause ocular dryness by inhibiting their secretion is not known. To perform these and other studies of pharmacological interest, we evaluated several micromethods for the analysis of tear samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, lactoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 microliters of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase assays based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or nylon membranes, prepared by an ordinary paper punch, which were coated with PGM and stained by Alcian blue or the periodic acid Schiff's reagent. The densitometric analysis was carried out using an ordinary flat scanner controlled by a personal computer equipped with an inexpensive software. The sensitivity of these simple assays was low (100-500 micrograms) but considered sufficient for certain studies. A more sensitive assay (5-20 micrograms) was developed by immobilizing PGM in small agarose gels (100 microliters), prepared in the wells of 96-well microplates, which could by stained by stains-all and analyzed by an automatic plate reader at 595 nm.

Hyaluronic acid (HA) is known to increase the ocular bioavailability of ophthalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require considerable amounts of mucin (M) that are difficult to obtain from ocular surfaces. Thus, we developed an alternative method, based on gel permeation liquid chromatography, to examine the interaction of HA with microgram quantities of mucin. HA (from human umbilical cord or rooster comb) were fractionated using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay based on the histological dye Stains-all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bound, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoadhesion of HA could be obtained. This method could be useful to study the interaction between HA and microgram quantities of ocular M (mucin), obtained from individual patients or normal subjects.

Natural hydrophobic substances like bile salts (cholate, deoxycholate, chenodeoxycholate, lithocholate and their conjugates with glycine and taurine), fatty acids (caprylic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acid) were much more active (EC50 approximately 10(-4)-10(-5) M) than selected amino acids (EC50 > 10(-2) M) and inorganic salts (EC50 approximately 10(-1) M) in inhibiting heat-induced denaturation of human serum albumin in vitro. Fish oil, rich in n-3-polyunsaturated acids such as eicosapentaenoic acid and docosahexaenoic acid, administered p.o. (1 ml/kg) in the rat, protected ex vivo (after 2 hr) serum against heat-induced denaturation more than bendazac, a known antidenaturant drug. Thus, we speculated that the antidenaturant activity of fish oil may be partly (in addition to the known effect on endogenous eicosanoid composition) responsible for its beneficial effects in rheumatoid arthritis and other rheumatic conditions. In this connection, it is of note that the in vitro antidenaturant activity of fish oil fatty acids was higher than that of known antidenaturant drugs such as bendazac and bindarit and nonsteroidal anti-inflammatory drugs like phenylbutazone and indomethacin which could exert beneficial effects in chronic inflammatory conditions by stabilizing endogenous proteins.

Compounds capable of inhibiting protein aggregation may find pharmacological applications in the treatment of a number of diseases called protein condensation diseases [Benedek (1997)], which include cataract, biliary and urinary lithiasis and certain rheumatic diseases. We examined the effect of selected compounds on heat-induced aggregation human serum albumin (HSA), IgG and lysozyme. HSA (0.2% w/v in 0.066 M sodium phosphate pH 5.3 at 22 degrees C), IgG (0.5% w/v in 0.066 M Tris pH 8.0 at 22 degrees C), and L (0.2 % w/v in 0.066 M CAPS pH 11.0 at 22 degrees C) were heated for 30 min at 70 degrees C in the presence or absence of different concentrations of the substance under examination and heat-induced aggregation of 100 microl aliquots was evaluated by measuring the absorbance at 595 nm using an automatic microplate reader. In these conditions, inhibition of aggregation could be due to an anti-denaturant effect or to interferences with the aggregation of denatured molecules, as previously described [Saso, Casini et al. (1998)]. However, this distinction may not be pharmacologically relevant when the target of the therapy is the prevention of abnormal phenomena of protein aggregation. Inorganic salts like NaCl and CaCl2 were active on the three proteins (IgG > HSA > L) but many ligands of HSA such as tryptophan, N-acetyl-tryptophan, caprylic acid, capric acid, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid and bendazac were active on their carrier but not on IgG and L, indicating that the latter proteins are more difficult to protect and that specific anti-denaturant and/or anti-aggregant compounds should be developed.

Preparative affinity chromatography of bovine serum amine oxidase (SAO) on aminohexyl (AH)-Sepharose was often associated with an unexpected irreversible SAO retention on the support. This particular enzyme immobilization, occurring without coupling reagents, was supposed to be due to a SAO ability to: (i) recognize alkylamine groups of the support as macro-molecularized substrate; (ii) catalyse their oxidation to the corresponding aldehydes, with release of NH3 and H2O2; and (iii) be immobilized on the activated support by a coupling between the nascent aldehyde groups and SAO free amine groups. This affinity immobilization procedure, with the self-activation of the support, being mild, allows by simple incubation for 24 h, the enzyme immobilization with the retention of 80% from original specific activity of free SAO. Immobilized SAO on AH-Sepharose microcolumns, viewed as a continuous flow-system reactor, was able to catalyse benzylamine oxidation for several weeks.