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Background: Efficacy of melatonin in treating sleep disorders has been demonstrated in numerous studies. Being with short half-life, melatonin needs to be formulated in extended-release tablets to prevent the fast drop of its plasma concentration. However, an attempt to mimic melatonin natural plasma levels during night time is challenging., Methods: In this work, Artificial Neural Networks (ANNs) were used to optimize melatonin release from hydrophilic polymer matrices. Twenty-seven different tablet formulations with different amounts of hydroxypropyl methylcellulose, xanthan gum and Carbopol®974P NF were prepared and subjected to drug release studies. Using dissolution test data as inputs for ANN designed by Visual Basic programming language, the ideal number of neurons in the hidden layer was determined trial and error methodology to guarantee the best performance of constructed ANN., Results: Results showed that the ANN with nine neurons in the hidden layer had the best results. ANN was examined to check its predictability and then used to determine the best formula that can mimic the release of melatonin from a marketed brand using similarity fit factor., Conclusion: This work shows the possibility of using ANN to optimize the composition of prolonged-release melatonin tablets having dissolution profile desired.

Objectives: A number of studies suggest that postprandial hyperglycemia produces oxidative stress, leading to complications associated with diabetes. However, hyperglycemia-induced oxidative stress may affect groups of people other than diabetics, such as smokers and athletes with specific diet plans. Based on previous reports that seated breathing meditation reduces hyperglycemia, the present study was designed to determine the effects of diaphragmatic breathing on postprandial plasma glycemia, insulin, oxidative stress, and antioxidant levels in athletes with normal glucose metabolism., Design: Data collected before and after consumption of a 900-calorie breakfast composed of 80% carbohydrates, 10% proteins, and 10% lipids were analyzed. Ten (10) minutes after the meal, 8 subjects spent 40 minutes performing diaphragmatic breathing in a quiet place. The other 8 subjects, representing the control group, spent the same time sitting in an equivalent quiet place reading a magazine., Subjects: Data from 16 amateur male cyclists age 30.12±4.9 years (±SD) were analyzed. Their mean height and weight were 177.81±5.3 cm and 71.40±5.2 kg, respectively. All subjects underwent a physical examination and were determined to be in good health., Outcome Measures: Blood samples were collected immediately before the meal as well as 1 hour and 2 hours after the meal, and plasma levels of glucose, insulin, reactive oxygen metabolites, and biologic antioxidant potential were determined. Heart rate was also recorded., Results: Results show that in normal subjects, acute hyperglycemia induces free-radical production while reducing the antioxidant levels (p<0.05). Diaphragmatic breathing reduces heart rates (p<0.01), increases insulin (p<0.05), reduces glycemia (p<0.01), and reduces free-radical production as indicated by the higher antioxidants levels (p<0.05)., Conclusions: Diaphragmatic breathing, likely through the activation of the parasympathetic nervous system, increases insulin, reduces glycemia, and reduces reactive oxygen species production.

Aim: We determined the combined effects of cold and exercise on oxidative stress during submaximal exercise., Methods: Sixteen amateur male cyclists pedaled at a constant speed corresponding to 85% of maximal HR as determined in normal conditions. Eight athletes pedaled indoors at 23 °C while 8 athletes pedaled outdoors at a temperature of 4-6 °C. We then evaluated the levels of reactive oxygen metabolites and plasma levels of antioxidants after exercise., Results: Performing a physical task in cold conditions increased the free radical production, as demonstrated by the augmented levels of reactive oxygen metabolites and the concomitant decrease of plasma levels of antioxidants in outdoors cyclists as compared to indoors cyclists. The overall ANOVA and the post-hoc comparisons revealed a significant exercise and temperature effect. The mean level of reactive oxygen metabolites in athletes who exercised indoors was significantly lower than that of the outdoor athletes. Moreover, the outdoors group presented plasma levels of antioxidants significantly lower than those of the indoors group., Conclusion: Since several sports are performed outdoors during the winter season, the increased risk of oxidative stress in cold conditions must be considered in these disciplines. Cyclists, football and rugby players, and runners are all affected by the elevation in oxygen radicals induced by cold and should take appropriate precautions, such as specific antioxidant integration.

The aim of this study was to evaluate the effect of Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502 on oxidative stress in athletes during a four-week period of intense physical activity. Two groups of twelve subjects each were selected for this analysis. The first group consumed a daily dose of a mixture of the two probiotic strains (1:1 L. rhamnosus IMC 501 and L. paracasei IMC 502; ~10(9) cells/day) for 4 weeks. The second group (control) did not consume any supplements during the 4 weeks. Blood samples collected immediately before and after the supplementation were analyzed, and plasma levels of reactive oxygen metabolites and biological antioxidant potential were determined. Faeces were also collected and analyzed before and at the end of the probiotic supplementation. Antioxidative activity and oxidative stress resistance of the two strains were determined in vitro. Results demonstrated that intense physical activity induced oxidative stress and that probiotic supplementation increased plasma antioxidant levels, thus neutralizing reactive oxygen species. The two strains, L. rhamnosus IMC 501(®) and L. paracasei IMC 502(®), exert strong antioxidant activity. Athletes and all those exposed to oxidative stress may benefit from the ability of these probiotics to increase antioxidant levels and neutralize the effects of reactive oxygen species.

Diaphragmatic breathing is relaxing and therapeutic, reduces stress, and is a fundamental procedure of Pranayama Yoga, Zen, transcendental meditation and other meditation practices. Analysis of oxidative stress levels in people who meditate indicated that meditation correlates with lower oxidative stress levels, lower cortisol levels and higher melatonin levels. It is known that cortisol inhibits enzymes responsible for the antioxidant activity of cells and that melatonin is a strong antioxidant; therefore, in this study, we investigated the effects of diaphragmatic breathing on exercise-induced oxidative stress and the putative role of cortisol and melatonin hormones in this stress pathway. We monitored 16 athletes during an exhaustive training session. After the exercise, athletes were divided in two equivalent groups of eight subjects. Subjects of the studied group spent 1 h relaxing performing diaphragmatic breathing and concentrating on their breath in a quiet place. The other eight subjects, representing the control group, spent the same time sitting in an equivalent quite place. Results demonstrate that relaxation induced by diaphragmatic breathing increases the antioxidant defense status in athletes after exhaustive exercise. These effects correlate with the concomitant decrease in cortisol and the increase in melatonin. The consequence is a lower level of oxidative stress, which suggests that an appropriate diaphragmatic breathing could protect athletes from long-term adverse effects of free radicals.

Aim: Ultimate is a sport played by hundreds of thousands of people in more than 42 countries; however, it is still mainly known as a recreational more than a team sport, and further studies are needed to define its physical load. Particularly, since no studies relating Ultimate to hydration have been performed, we aimed to determine body fluid balance, voluntary water intake and the most reliable method for assessing the hydration status of players after a typical 80-minute Ultimate match., Methods: bioimpedance, urine specific gravity and body mass changes to asses the hydration level of the players were measured., Results: It was observed that not all of the methods are adequate to determine dehydration in Ultimate players, and that measurement of body mass changes represents a reliable and accurate technique., Conclusions: These findings demonstrate that ultimate as an intense sport that can induce significant fluid loss, which is not always replaced by individual drinking.

Doxazosin analogues 1-3 and 1a were synthesized and investigated at alpha1-adrenoceptors and PC-3, DU-145, and LNCaP human prostate cancer cells. Compound 1 (cyclodoxazosin) was a potent alpha(1B)-adrenoceptor antagonist displaying antiproliferative activity higher than that of doxazosin in cancer cells in vitro and in vivo, respectively. Because of its antitumor efficacy at low concentrations, lower apoptotic activity in NHDF vs tumor cells, and antiangiogenetic effect, 1 showed a better therapeutic profile relative to doxazosin.

Aim: This work monitored changes in oxidative stress and antioxidant defence during an endurance exercise in over 40 years old athletes., Methods: Subjects were monitored during the 24-hours mountain bike Idro Lake (North of Italy) competition which took place in June 2008. The race lasted for 24 h, starting at 10.00 a.m., ending at 10.00 a.m. of the following day and was based upon riding for as many kilometers as possible in the 24-hours time schedule in a 5.5 km circuit trail. The study included 6 men bikers, aged 44.8 +/- 2 years, who raced on an individual basis. Blood samples were collected and the oxidative stress was measured performing the d-ROMs test which determined the reactive oxygen metabolites (ROMs), whereas the antioxidant defence status was assessed determining the biological antioxidant potential (BAP test)., Results: The ROMs levels significantly increased after 8 h from the beginning of the competition (122 %), at the end of the race (162%), 24 h (158%) and 48 h (144%) post-race. The biological antioxidant potential significantly increased at the end of the race (128%) and remained elevated 48 h later (114%). After 72 h post-race, ROMs and BAP levels differed significantly amongst subjects, thus showing an individual response to oxidative stress., Conclusions: In conclusion, exposure to intense and prolonged exercise induced a marked increase in dROMs levels in master athletes, only partially counterbalanced by antioxidants in blood plasma. The long-term effects of oxidative agents on the human body requires further studies, but it is likely that a diet potentially rich in antioxidants would help preventing oxidative damage of body cells and tissues and enhancing recovering from the endurance performance.

Background: In this study, we investigated the effect of CRM197 treatment in human adrenocortical carcinoma (AC) implanted in nude mice. CRM197 is a non-toxic mutant of diphtheria toxin that binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) which is implicated in the proliferative activity of several tumor cells., Methods: HB-EGF expression in AC cells was evaluated by reverse transcription PCR and Western blot. AC tumors were implanted in nude mice and then treated with CRM197. Effects of treatment on angiogenesis and apoptosis were investigated by immunohistochemistry and Western blot. The effects on cell invasion and migration were investigated with a matrigel invasion assay., Results: We demonstrated that human AC cells express HB-EGF. A treatment with CRM197 blocked growth, reduced angiogenesis and induced apoptosis in AC tumors implanted in nude mice. CRM197 also inhibited invasion and migration of these tumor cells., Conclusions: These data support the evidence for anticancer properties of CRM197 in AC tumors., (Copyright 2009 S. Karger AG, Basel.)

Background: Since obesity and related diseases are now considered epidemic, new and more accurate formulas for epidemiological studies are of interest to the scientific community. Several equations have been proposed to estimate the body composition simply from anthropometric measurements. However, with time, the body composition of the populations studied changes in relation to their food habits and lifestyle, and, therefore, the equations must be regularly updated and corrected., Aim of the Study: The aim of the study was to develop new equations to determine the body composition among the Italian population using the body mass index and independently by variables such as age and body structure., Methods: Bioelectrical impedance and anthropometric analysis of 764 Italian Caucasian subjects (342 females and 422 males), 11 to 80 years of age, were analysed. Females and males were analysed separately. Multiple regression analyses were performed in order to estimate the body composition of the subjects. The estimated masses were then compared with the measured masses using Bland and Altman plots. We also calculated the differences between the estimated and measured masses, reported as % of the body weight, for the 95, 85 and 75 degrees percentile of the female and male groups. Finally we compared our formulas with the Watson equations, which are used to estimate the total body water., Results: All body masses estimated were positively correlated to the measured values. Moreover, at any percentile analysed, our formulas resulted more precise than the Watson formula. Equations: Females: FM = 1.9337 BMI - 26.422; FFM = BW - FM; BCM = 0.3655 FFM + 4.865; TBW = 0.5863 FFM + 7.1732; Males: FM = 1.407 BMI - 21.389; FFM = BW - FM; BCM = 0.4485 FFM + 3.3534; TBW = 0.6997 + 1.4567., Conclusions: Although an inevitable inaccuracy must be expected in epidemiological studies, our equations are adequate to analyze the body composition state and changes occurring among the Italian population by simply considering weight and height.

Adrenocortical carcinoma is a rare tumor of the adrenal gland which requires new therapeutic approaches as its early diagnosis is difficult and prognosis poor despite therapies used. Recently, mebendazole has been proved to be effective against different cancers. The aim of our study was to evaluate whether mebendazole may result therapeutically useful in the treatment of human adrenocortical carcinoma. We analyzed the effect of mebendazole on human adrenocortical carcinoma cells in vitro and after implantation in nude mice. In order to clarify mechanisms of mebendazole action, metastases formation, apoptosis and angiogenesis were also investigated. Mebendazole significantly inhibited cancer cells growth, both in vitro and in vivo, the effects being due to the induction of apoptosis. Moreover, mebendazole inhibited invasion and migration of cancer cells in vitro, and metastases formation in vivo. Overall, these data suggest that treatment with mebendazole, also in combination with standard therapies, could provide a new protocol for the inhibition of adrenocortical carcinoma growth.

Background: Malignant pleural mesothelioma (MPM) is a tumor known to be resistant to conventional therapies. Thus, an in vivo model can represent an important tool for assessing the efficacy of novel approaches in the treatment of MPM.Presently, human MPM cells have been grown orthotopically in mice upon transplantation of tumor masses or tumor cell suspensions following surgery. In these models however, surgery can interfere with the tumor growth and the early stages of tumor development cannot be easily explored. Finally, results may not be so accurate due to implantation of potentially different tumor samples in different experimental groups.Our work aimed at establishing a nude mouse model xenotransplanted with human MPM cell lines in which tumor progression exhibits some features of the human disease., Methods: Three distinct human MPM cell lines previously established from MPM patients displaying two different phenotypes, biphasic (MM-B1 and IST-Mes3) and epithelioid (IST-Mes2), were directly injected into the pleural cavity of nude mice. At different times, mice were sacrificed for autopsy, tumor nodules were counted and then removed for histology. Presence of metastases in visceral organs was also monitored., Results: IST-Mes2 cells were unable to grow in nude mice. MM-B1 and IST-Mes3 cells were capable of growing in nude mice and formed tumor nodules in the pleura. Post-mortem examination showed that MPM cells progressively colonized the parietal and visceral pleura, the diaphragm, the mediastinum and, lastly the lung parenchyma. No pneumo-thorax was evidenced in the mice. Pleural effusions as well as lymph node metastases were observed only at later times., Conclusion: This model mimics the progression of human malignant mesothelioma and it is easy to perform and reproducible; therefore it can be useful to study human MPM biology and evaluate the efficacy of novel therapies.

Losartan, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'(1H-tetrazol-5-yl)-biphenil-4-yl)methyl]imidazole, and Irbesartan, 2-n-butyl-3-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one, are two angiotensin AT1 receptor antagonists largely used in human health care as antihypertensive agents. Their ability to cross the blood-brain barrier and to influence the central renin-angiotensin system are widely investigated, but how this brain system responds to the subchronic and chronic block of the angiotensin AT1 receptor is still unknown. Normotensive rats were intragastrically implanted for 7- and 30-day administration, with a dose of 3 and 30 mg/kg body weight. Treatments were shown to influence, in a dose-, time- and brain-area-dependent manner, angiotensinogen mRNA levels in scanned areas. This study showed a general up-regulation of angiotensinogen mRNA expression after 7 days and a widespread down-regulation or basal level of expression after a 30-day administration of two angiotensin AT1 receptor antagonists.

The antiproliferative effect of serotonin-reuptake inhibitors (SSRI) and serotonin antagonists has been demonstrated in prostate tumors. Since Hypericum perforatum components act as serotonin-reuptake inhibitors and exert cytotoxic effects on several human cancer cell lines, in this work we analyzed the effect of a treatment with Hypericum perforatum extract (HPE) on the growth of human prostate cancer cells in vitro and in vivo. This study highlighted a significant reduction of tumor growth and number of metastasis suggesting that this natural compound may be useful in the treatment of prostate cancer., (Copyright 2004 Elsevier Ireland Ltd.)

The aim of the present study was to investigate if aging is associated with platelet membrane modifications possibly related with cellular activation and hyperaggregability and if platelets from centenarians show different properties which might play a role in successful aging and longevity. Platelet plasma membranes were obtained from 60 healthy subjects, divided into four groups according to the age range: (1) 21-39 years; (2) 40-59 years; (3) 60-79 years; (4) centenarians (>/=100 years). Both centenarians and control subjects were submitted to the following inclusion criteria: liver, kidney, and thyroid function tests within the normal range; absence of history of diabetes, hypertension or coronary heart disease; no signs of edema or dehydration; no drug or vitamin supplement in the 4 weeks before the study; absence of Alzheimer's disease or secondary dementia. The following determinations were performed: lipid peroxide levels (Lp) evaluated by the measurement of thiobarbituric acid (TBA) reactivity, fluidity studied by the fluorescence anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), Na(+)/K(+)-ATPase activity measured by the method of Kitao and Hattori, and sialic acid (SA) content evaluated by the periodate-thiobarbituric acid method. Centenarians showed: (i) Lp concentrations lower than elderly subjects; (ii) increased Na(+)/K(+)-ATPase activity compared with adult and elderly subjects; (iii) higher TMA-DPH anisotropy than elderly subjects; (iv) SA content similar to the young and adult groups.The present work found deep platelet membrane modifications in centenarians compared with elderly subjects. These changes are likely associated with a decreased platelet activation and therefore might exert a protective role against cardiovascular accidents, as platelet activation is a key event in the initiation and progression of arteriosclerosis.

This study tested the effects of 8 days of subchronic administration of 3,4-methylene dioxymethamphetamine (MDMA) (5 mg/kg b.w.) on preprotachykinin A mRNA levels in discrete rat brain regions. In situ hybridization examined preprotachykinin A mRNA levels in the core and shell of the nucleus accumbens, the islands of Calleja, the olfactory tubercle, the dorsal and ventral caudate-putamen, the bed nucleus of the stria terminalis, the medial preoptic area, the medial habenular nucleus and in the postero-dorsal part of the medial amygdala. Higher levels of preprotachykinin A mRNA were found in the core and shell of the nucleus accumbens, in the islands of calleja, in the olfactory tubercle, in the bed nucleus of the stria terminalis, in the medial habenular nucleus and the postero-dorsal part of the medial amygdala, compared to control animals. Conversely, increased preprotachykinin A mRNA levels were observed in the dorsal and ventral caudate-putamen in MDMA treated when compared to control rats. In the social memory test, MDMA significantly impaired rats' short-term working memory. These results show that chronic exposure to MDMA strongly affects preprotachykinin A mRNA levels in discrete rat brain regions. These changes occur in experimental conditions in which working memory is markedly reduced, suggesting that changes in gene expression of tachykinin mechanisms may contribute to the effects of MDMA on memory function.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

Aims/hypothesis: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects., Methods: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 micromol/l homocysteine. Stimulation experiments were done in vitro by the addition of alpha-thrombin (0.2 U/ml)., Results: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects., Conclusion/interpretation: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation.

Decreased levels of nitric oxide (NO) may contribute to impaired endothelium-dependent vasodilatation in essential hypertension. Moreover, in hypertension, major platelets aggregation and endothelial adhesion, and increased atherogenetic risks are also present. Nitric oxide produced by platelet NO synthase, which is similar to endothelial NO synthase, inhibits platelets aggregation by increasing cytoplasmic cyclic GMP levels and contributes in a major way to the antithrombogenic properties of endothelium. The aim of this study was to investigate platelet NO production and cytosolic Ca2+ levels in patients with essential hypertension and in healthy subjects. We studied NO production in 36 subjects (21 patients had essential hypertension and 15 subjects were normotensive); NO synthase activity was evaluated by measuring nitrite levels by the Griess reaction in the supernatant of sonicated platelets. Cytosolic Ca2+ levels were measured in intact platelets using the fluorescent probe Fura 2-AM. Nitric oxide levels in platelets were found higher in normotensive than in hypertensive patients (P < .0001). Nitric oxide levels in hypertensive women were significantly higher than in hypertensive men (P < .001). Hypertensive women and men had lower levels of nitrite than normotensive women and men (P < .001 and P < .002, respectively). Platelet cytosolic Ca2+ levels were higher in hypertensive patients than in normotensive subjects (P < .001). An inverse correlation was found between platelet cytosolic Ca2+ and NO levels (r = 0.74, P < .002). These data confirm the link between hypertension and altered platelets function and suggest a role for NO in cardiovascular events. Moreover, the higher levels of nitric oxide in child-bearing age women than in men further support the protective effect of estrogens on cardiovascular diseases.

Human whole saliva contains a number of antimicrobial agents, and lysozyme, lactoferrin, secretory IgA and peroxidase are among the best known. Peroxidase catalyzes a reaction involved in the inhibition of bacterial growth and metabolism, and the prevention of hydrogen peroxide accumulation, thus protecting proteins from the action of oxygen and reactive oxygen species (ROS). To better understand the role played by the oxidative stress in the aging process, we studied the relationship between total protein content, peroxidase activity, malondialdehyde (MDA) and nitric oxide (NO) content of human unstimulated whole saliva in 169 healthy subjects subdivided into groups according to age. Our results show a significant decrease in peroxidase activity with age. Moreover, the increase in saliva lipid peroxide levels indicates an enhanced free radical production that may contribute to tissue damage. On the other hand, findings concerning human unstimulated whole saliva NO content showed a significant increase in elderly subjects, suggesting that an enhanced NO production might depend on a stimulation of leukocyte-inducible NO synthase (i-NOS) activity. Our results suggest that during aging the oral tissues may become more susceptible to environmental factors due to a modification in the balance between different antimicrobial agents.

The direct and indirect interaction between the nervous system and its transmitters with the immune system was evaluated in the rat by using the neurotoxin capsaicin (Caps). In the present study we investigated the effect of Caps administration to neonatal rats on thymocyte subpopulation distribution and functions at different times after treatment. Caps treatment results in a marked reduction of thymus weight and cellularity. As shown by immunofluorescence and FACS analysis, profound depletion of double negative (DN), double positive (DP), and single positive (SP) CD4(+) cells was already evident at day 7 after treatment and persisted until day 28. Reduced numbers of SP CD8(+) cells were observed only at later time points. Analysis of TCR phenotype indicates that CD5(+) TCR gamma/delta(+) are particularly sensitive to neonatal Caps treatment. Caps-induced thymocyte depletion was associated with reduced proliferation in response to T cell mitogens. Moreover, in situ TUNEL reaction and agarose gel electrophoresis indicate that neonatal Caps treatment induces apoptosis of thymus cells. Morphological analysis reveals the presence of apoptotic cells in the subcapsular thymus cortical region. Overall our results suggest that Caps when administered at birth, profoundly affects T cell differentiation, likely through its ability to activate apoptotic cell death program.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

The aim of the present study was to evaluate the action of plasma from insulin-dependent diabetic (IDDM) pregnant women on nitric oxide synthase (NOS) activity in cultured human umbilical vein endothelial cells (HUVECs). We also studied the effect of the plasma on cytosolic calcium and on Na+/K+-adenosine triphosphatase (ATPase) activity. Dynamic fluorescence studies of membrane fluidity were contemporarily performed to detect a direct effect of plasma on the endothelial cell membrane. We observed a significant increase in NOS activity, intracellular calcium, and Na+/K+-ATPase activity in cultured HUVECs exposed to IDDM plasma. Our dynamic fluorescence study showed a different microenvironmental organization of the cellular membrane after incubation with plasma from IDDM pregnant women, with a marked decrease in microheterogeneity as evaluated in terms of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) lifetime distribution width. The present investigation suggests that plasma from IDDM pregnant women can cause a generalized disturbance in the function of endothelial cells cultured from healthy subjects. Such a modification might play a central role in the pathogenesis of the vascular complications of the disease.

The aim of this study was to evaluate changes in the concentration of certain components of human unstimulated whole saliva during aging, in order to better understand the role played by aging in oral health. In particular, we studied total protein concentration, alpha-amylase activity, sialic acid content and calcium and phosphorus concentrations in 100 healthy subjects of both genders, aged between 10 and 80 years, who were subdivided into four groups according to their age: 10-25 years, 26-40 years, 41-65 years, and 66-80 years. Other than sialic acid, the concentrations of the components studied were not affected by age. There was a significant negative correlation between sialic acid content and age. Our data indicate the presence of a decreased submandibular/sublingual function with aging, thus suggesting the possibility of a concomitant reduction in the modulating action of unstimulated whole saliva on the oral flora.

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

Sialic acid (SA) content and Na+/K+-ATPase activity of red blood cell (RBC) membranes were studied in 26 normoalbuminuric patients with insulin-dependent diabetes mellitus (IDDM), 25 normoalbuminuric patients with non-insulin-dependent diabetes mellitus (NIDDM), and 40 healthy nondiabetic subjects with a negative family history for diabetes. A decrease in RBC membrane SA content and Na+/K+-ATPase activity was observed in older control subjects compared with younger controls. A significant correlation between age, Na+/K+-ATPase activity, and SA content was also found. No difference was observed in RBC membrane SA content between IDDM and NIDDM subjects, but Na+/K+-ATPase activity was significantly lower in IDDM patients. SA content was increased in NIDDM subjects compared with healthy subjects of similar age, whereas Na+/K+-ATPase activity was significantly lower in both IDDM and NIDDM subjects compared with controls. In NIDDM, Na+/K+-ATPase activity was significantly correlated with age, whereas both Na+/K+-ATPase activity and SA content were significantly correlated in IDDM and NIDDM patients. Hemoglobin A1c, (HbA1c) levels did not show any significant correlation either with Na+/K+-ATPase or with SA content in diabetic patients. The modified SA content and Na+/K+-ATPase activity in elderly subjects described in the present study indicate a similar behavior of the erythrocyte membrane during both RBC senescence and aging of subjects.