Lipid patterns in the saliva of smoking young adults.

Salivary lipids are important for the maintenance of oral cavity health. Elevated salivary lipid levels are associated with an increase of caries incidence, plaque development, calculus formation and periodontal disease. However, the regulation of lipid salivary levels is scarcely known. Cigarette smoke is considered a risk factor for oral cavity diseases. We study how cigarette smoke may affect the secretion of salivary lipids. To this purpose, we determine the salivary levels of cholesterol and of glycerolipids in saliva sampled from smokers and non-smokers at various times of day. We observe an increase of glycerophospholipid and a decrease of cholesterol levels in the smokers' saliva collected at 10 p.m. On the other hand, unsaturated fatty acids in chief phospholipids of saliva are lower in smokers at 7 a.m. Therefore, for the first time, we demonstrate that cigarette smoke induces variations of saliva lipid pattern in young people even moderately smoking.

Micropatterned hydrogenated amorphous carbon guides mesenchymal stem cells towards neuronal differentiation.

This study investigated how the design of surface topography may stimulate stem cell differentiation towards a neural lineage. To this end, hydrogenated amorphous carbon (a-C:H) groove topographies with width/spacing ridges ranging from 80/40µm, 40/30µm and 30/20µm and depth of 24 nm were used as a single mechanotransducer stimulus to generate neural cells from human bone marrow mesenchymal stem cells (hBM-MSCs) in vitro. As comparative experiments, soluble brain-derived neurotrophic factor (BDNF) was used as additional biochemical inducer agent. Despite simultaneous presence of a-C:H micropatterned nanoridges and soluble BDNF resulted in the highest percentage of neuronal-like differentiated cells our findings demonstrate that the surface topography with micropatterned nanoridge width/spacing of 40/30µm (single stimulus) induced hBM-MSCs to acquire neuronal characteristics in the absence of differentiating agents. On the other hand, the alternative a-C:H ridge dimensions tested failed to induce stem cell differentiation towards neuronal properties, thereby suggesting the occurrence of a mechanotransducer effect exerted by optimal nano/microstructure dimensions on the hBM-MSCs responses.

Erectile dysfunction and NO: variations of NO penile blood levels before and after sildenafil treatment.

Erectile dysfunctions are not uncommon, especially in patients suffering from metabolic syndrome and from a number of circulatory and psychiatric problems. cGMP diesterase inhibitors, such as sildenafil, have proven to be beneficial in the treatment of many such conditions. Our patients, all of them complaining of erectile dysfunction, were treated with sildenafil (50 mg, thrice a week for 6 weeks). All patients reported beneficial effects and were not clinically distinguishable (interview and Doppler scores). We sampled blood for systemic circulation (cubital vein) and from penis (corpora cavernosa) before and after prolonged sildenafil treatment, and measured nitrate (+nitrite) levels in plasma and in red blood cells (RBCs). Hemoglobin is a powerful catalyst of NO oxidation to nitrate, and we thought that nitrate in RBC might be a more sensitive parameter than plasma nitrate. We found that the ratio of penile vs systemic blood plasma nitrate was similar in all patients before or after sildenafil treatment. On the other hand, the same parameter measured in RBC showed that, at the beginning of treatment, patients could be divided into two groups: one with a high ratio and the other with a low ratio. Therefore, clinically similar patients could be biochemically divided into two populations. The difference disappeared after treatment, thus hinting at a curative effect of the drug. The mechanisms underlying this behavior are still unknown and the clinical implication of two populations that can be distinguished by RBC nitrate is yet to be evaluated.

Recovery of sexual function after nerve-sparing radical retropubic prostatectomy: is cavernous nitric oxide level a prognostic index?

The preservation of NANC nerve fibers (producing nitric oxide, NO) is necessary for erection recovery after retropubic radical prostatectomy (RRP). Yet, it is impossible to establish when and if a patient will recover erections; therefore, we investigate the prognostic value of cavernous blood NO levels on this parameter. Nerve-sparing RRP was performed on 14 patients for localized prostate cancer. We evaluated all patients 3 months after surgery by IIEF score: no patients had erections. A cavernous blood sample was also taken to determine NO levels (as nitrite). Patients were evaluated again 18 months after surgery. In six cases, erectile function was compromised, whereas in seven cases, potency was restored. Statistical analysis showed a relationship between nitrite levels in cavernous blood 3 months after surgery and the recovery or erectile function at 18 months. We propose that cavernous NO blood levels are a prognostic index of erection recovery.

No hemoglobin but NO: the icefish (Chionodraco hamatus) heart as a paradigm.

The role of nitric oxide (NO) in cardio-vascular homeostasis is now known to include allosteric redox modulation of cell respiration. An interesting animal for the study of this wide-ranging influence of NO is the cold-adapted Antarctic icefish Chionodraco hamatus, which is characterised by evolutionary loss of hemoglobin and multiple cardio-circulatory and subcellular compensations for efficient oxygen delivery. Using an isolated, perfused working heart preparation of C. hamatus, we show that both endogenous (L-arginine) and exogenous (SIN-1 in presence of SOD) NO-donors as well as the guanylate cyclase (GC) donor 8Br-cGMP elicit positive inotropism, while both nitric oxide synthase (NOS) and sGC inhibitors, i.e. L-NIO and ODQ, respectively, induce significant negative inotropic effects. These results therefore demonstrate that under basal working conditions the icefish heart is under the tonic influence of a NO-cGMP-mediated positive inotropism. We also show that the working heart, which has intracardiac NOS (shown by NADPH-diaphorase activity and immunolocalization), can produce and release NO, as measured by nitrite appearance in the cardiac effluent. These results indicate the presence of a functional NOS system in the icefish heart, possibly serving a paracrine/autocrine regulatory role.

Role of human prostasomes in the activation of spermatozoa.

Prostasomes are small vesicles of prostatic origin contained in human semen. Their composition is peculiar under many aspects. Cholesterol is abundant and many proteins are endowed with enzymatic or other activities. The function of prostasomes has been amply debated and several hypotheses have been put forward. The liquefaction of semen, spermatozoa motility, antibacterial activity and immunological functions have been related to prostasomes. Under certain aspects, prostasomes resemble synaptosomes. The fusion of prostasomes to spermatozoa enriches spermatozoa with cholesterol and causes bursts of cytoplasmic sperm calcium. The interaction of spermatozoa and prostasomes should be limited to vagina since prostasomes are immobile and do not follow spermatozoa in the superior female genital tract. Calcium bursts would increase spermatozoa motility, where cholesterol would decapacitate spermatozoa, so preventing untimely activation. Since spermatozoa receive many different molecules from prostasomes, additional effects are also possible. Prostasomes makes spermatozoa more apt to be activated by progesterone in the proximity of the ovum. Therefore, the fusion between spermatozoa and prostasomes would influence spermatozoa behaviour under many aspects and might be relevant for fecundation. The richness of molecular species in prostasomes is amazing and these small vesicles are expected to lead to many more discoveries in the field of human reproduction.

NO synthesis in human saliva.

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria (S. salivarius, S. mitis and S. bovis) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.

Hydrodynamic radii and lipid transfer in prostasome self-fusion.

The characteristics of human prostasomal vesicles have been investigated by three methods, namely, dynamic light scattering, transfer of a lipophylic fluorescent dye (R18), and electron microscope appearance. The vesicle preparations were stable for a long time and their diameters were in the range of 200 nm. The exposure to acidic pH values (about 5) increased both particle radii and the transfer of R18. The microscopic appearance was also affected by the pH value. We infer that these changes are due to a self-fusion of prostasome vesicles; this fusion is protein-dependent since various methods used by us and able to affect the protein component of membranes (boiling, extraction of lipid and liposome preparation, treatment with pronase) all abolished the effect seen at pH 5 on intact particles.

Reaction mechanism between nitric oxide and glutathione mediated by Fe(III) myoglobin.

Ferrimyoglobin at pH 7.4 binds nitric oxide to yield nitric oxide adducts. In the presence of glutathione (GSH), nitrosoadducts of Mb(III) react with it to give nitrosoglutathione, whose concentration has been determined with an apparatus based on a specific and sensitive solid-state amperometric gas sensor. The reaction constant between the adduct and glutathione, kGSH = (47 +/- 1) M(-1) x s(-1), obtained by UV-Vis spectroscopy kinetic measurements, is about one-eighth of the constant with OH- determined by other authors. We can explain this fact with the higher nucleophilicity of OH- compared to GSH, due to the bulkiness and charge of the species. It is known that the formation of nitrosothiols starting from nitrite or NO (nitrogen monoxide) and glutathione, in the absence of oxygen, is impossible. Thus, from a biological point of view, it is important to point out that GSH reacts with NO in the presence of ferrimyoglobin, even at physiological pH, to form nitrosoglutathione.

Progesterone-induced increase of sperm cytosolic calcium is enhanced by previous fusion of spermatozoa to prostasomes.

Ejaculated spermatozoa must undergo a number of modifications before becoming able to fertilize the oocyte. The interaction of sperm with other semen components may influence these phenomena; human semen contains vesicles of prostatic origin, called prostasomes that may fuse to sperm at slightly acidic to neutral pH values. Prostasomes contain calcium and it has been demonstrated that their fusion with spermatozoa produces a transient increase (wave) of [Ca(2+)](i) in these cells. The fusion process also transfers protein and lipid to spermatozoa. These phenomena may induce long-lasting changes of sperm properties. We test the hypothesis that spermatozoa, as modified by fusion, change their ability to undergo the progesterone-induced increase of [Ca(2+)](i) and we find that the increase of [Ca(2+)](i) produced by the fusion with prostasomes and by the stimulation with progesterone are independent and additive phenomena. We also find that spermatozoa present a stronger response to the progesterone-induced increase of [Ca(2+)](i) if they are previously made to fuse with prostasomes. This effect does not depend directly on the [Ca(2+)](i) increase due to fusion, since it is still present after the [Ca(2+)](i) has returned to resting values.

Lipid fatty acid and protein pattern of equine prostasome-like vesicles.

The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein pattern (SDS-PAGE electrophoresis) shows that the 30-50-kDa fraction is less abundant in stallion vesicles. In addition, fluidity (measured as fluorescence anisotropy of diphenylhexatriene) is higher in horse prostasome-like vesicles than in human prostasomes, albeit being much lower than that of most membranes. These findings may be connected to some species-related differences in reproductive physiology: the vaginal milieu of the mare is not acidic and the deposition of semen is intrauterine in the horse but vaginal in humans.

Determination of S-nitrosohemoglobin using a solid-state amperometric sensor.

Nitric oxide (NO, nitrogen monoxide), generated in biological systems, plays important roles as a regulatory molecule. Its ability to bind to hemoglobin (Hb) iron is well known. Moreover, it may lose an electron, forming the nitrosonium ion, involved in the synthesis of nitrosothiols (RSNO). It has been suggested that S-nitrosohemoglobin (SNO-Hb) may act as a reservoir of NO. The S-nitrosylation of Hb can be detected after the incubation of CysNO and Hb for 60 min with a molecular ratio (CysNO/hem) of 1:1. Upon increasing the ratio to 10:1, about 50% of total Hb (100% of beta-chain -SH 93) was derivatized in 60 min. In this paper, we describe a new method for the quantitative assay of SNO-Hb, after the liberation of NO by Cu(2+)/Cu(+) and the simultaneous assessment of NO by solid-state amperometric sensor. The assay described by us is sensitive, rapid, easy to perform, and inexpensive. For this reason, we believe that it may represent an important analytical improvement for the study of the S-transnitrosylation reactions between RSNO and the Hb Cys-beta 93 and SNO-Hb and glutathione.

Distribution of lipid and protein in human semen fractions.

Human semen is formed by the secretions of different glands. We fractionated semen by centrifugation and obtained four main fractions: (a) spermatozoa, (b) material precipitating at 10¿ omitted¿000xg, (c) prostasomes (precipitate at 105¿ omitted¿000xg), and (d) a soluble fraction. When required, fractions were purified further. We find that most semen protein (about 85%) is in the soluble fraction, 7% in spermatozoa and the remainder is scattered in the other fractions. We compared the electrophoretic pattern of soluble protein with the protein of prostasomes and found marked differences. On the other hand, prostasomes, that comprises only about 3% of total semen protein, contain about 45% of cholesterol and almost 15% of lipid phosphorus with a cholesterol to phospholipid molar ratio greater than 2. On the contrary, phospholipid is largely bound to the fraction containing spermatozoa (about 46% of total lipid phosphorus). This fraction is poor in cholesterol and has a cholesterol to phospholipid molar ratio of about 0.2. The distribution of lipid phosphorus among lipid classes shows some similarity in the soluble fraction and in prostasomes; in both fractions, sphingomyelin is the most abundant phospholipid (about 50%). On the other hand, phosphatidylcholine is the main phospholipid in spermatozoa-enriched fractions (about 35% of total lipid phosphorus). We conclude that the various fractions of seminal plasma obtained by centrifugation differ markedly from each other as to lipid and protein content.

Increase of human spermatozoa intracellular Ca2+ concentration after fusion with prostasomes.

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and calcium, and some of their proteins are enzymes. Prostasomes are involved in a number of biological functions. In previous work, we discovered that prostasomes may fuse to sperm at neutral or at slightly acidic pH values. This mechanism may deliver calcium to sperm, thereby influencing sperm functions. We measured sperm [Ca2+]i with the fura-2 AM method and found that it increased after mixing prostasomes and sperm at pH values allowing fusion (pH 5-7). The increase of [Ca2+]i was proportional to the extent of fusion as measured through the relief of R18 self-quenching. We also examined the increase of sperm [Ca2+]i and the extent of fusion as a function of sperm to prostasome ratio and, also in this case, there was proportionality between the extent of fusion and the increase of [Ca2+]i that reached its maximal values in about 10-20 min. However, a detectable increase of [Ca2+]i was attained after 2 min of fusion. This would represent a new mechanism to influence sperm [Ca2+]i besides ion-exchange systems and ATP-dependent pumps. The value of [Ca2+]i remained elevated, unless Na+ was also present in the external medium. Therefore, the mechanism of fusion might influence deeply the physiology of sperm by producing a transient increase of [Ca2+]i.

The motility of human spermatozoa as influenced by prostasomes at various pH levels.

Human semen contains several components among which spermatozoa, membranous vesicles called 'prostasomes', secreted by the prostate gland and unorganized material. Prostasomes possess an unusual lipid composition, contain a number of proteins and small molecules and have been claimed to take a part in the immune response, in seminal fluid liquefaction and in sperm motility. Since sperm may come in contact with an acidic environment in the vagina, it may be of some interest to know whether prostasomes may affect spermatozoon motility or may protect spermatozoa upon the exposure to an acidic milieu. Human semen was supplied by donors. From whole semen we collected spermatozoa by centrifugation and used the supernatant to prepare prostasomes (centrifugation at 105,000 g for 120 min, followed by purification step on Sephadex G 200); spermatozoa were then collected by a swim-up procedure and exposed to an acidic pH medium (from 5 to 7) in the presence or absence of prostasomes. Spermatozoa motility was subsequently assessed with a superimposed image analysis system (SIAS). Results indicate that the motility of spermatozoa was affected by the pH value of the medium. Acidic media reduced the percentage of motile cells and decreased the straight line velocity of spermatozoa (VLS). Prostasomes had a protective effect and increased the percentage of motile cells. However, they did not change the characteristics of motility (curvilinear and straight). Prostasomes may be considered as a system for counteracting the negative effects of acidic pH values that may be present in the vagina after coitus.

Interactions between prostasomes and leukocytes.

Prostasomes are membranous vesicles (150-200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and Ca2+. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated sperm and are involved in a number of biological functions. In a previous work, we found that prostasome can fuse to spermatozoa at slightly acidic pH values, as demonstrated by the transfer of the lipophilic octadecylrhodamine probe. In this paper, we study the interactions of two leukocyte populations (polymorphonuclear and mononuclear) with prostasomes and find a pH-dependent adhesion (revealed by microscopic observation), but no fusion. These phenomena may be relevant for the functions of leukocytes in human reproduction.

Fatty acid pattern of human prostasome lipid.

Prostasomes are organelles of prostatic origin found in human semen. Their average diameter is about 150 nm and they appear as a lipoprotein membrane surrounding less organized material. Their lipid composition is peculiar, having much cholesterol and sphingomyelin. On the other hand, many of their proteins possess catalytic activity and are involved in the immune response. In previous work, we have shown that prostasomes may fuse to sperm at slightly acidic pH values, thereby modifying the composition of the sperm plasma membrane. In this paper, we examine the fatty acid pattern of prostasome lipid and find that it is completely different from that of sperm membrane lipid. Polyunsaturated phosphatidylcholines, common in sperm membrane, are rare in prostasome. Therefore, the fusion between prostasomes and sperm should stabilize sperm plasma membrane by enriching it in cholesterol, sphingomyelin, and saturated glycerophospholipid. This would prevent the untimely occurrence of the acrosome reaction.

Prostasome-like particles in stallion semen.

Human semen contains membranous vesicles called prostasomes. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin, and Ca2+. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. No prostasome-like vesicles have been described in horse semen up to now. We have demonstrated the presence of prostasome-like vesicles in the equine semen and characterized them as to size, morphology, and lipid composition; we have found that they are similar to human prostasomes in many respects. We propose that these vesicles might be important for the fecundity of horse semen. This is of interest since the success of artificial insemination is limited by the fact that stallion sperm barely survive cryopreservation.

A new assay for the determination of low-molecular-weight nitrosothiols (nitrosoglutathione), NO, and nitrites by using a specific and sensitive solid-state amperometric gas sensor.

Nitric oxide (NO) is generated in biological systems and plays an important role as a bioregulatory molecule. Its ability to bind hemoglobin and myoglobin is well known. Moreover, it may lose an electron forming the nitrosyl group involved in the formation of S-nitrosothiols. The main problem in analyzing NO is its extreme reactivity. We have tackled this task by using an amperometric sensor to determine free NO, S-nitrosothiols (such as S-nitrosoglutathione), and nitrite in cell-free systems and murine microglial cell cultures. The determination of nitrosothiols is of biochemical relevance and a difficult task particularly at low concentration values. In this article we describe a new method based on the reductive cleavage of the S-NO bond by cuprous ions followed by a solid-state amperometric determination. The system described by us is sensitive, rapid, does not require previous purification steps, is easy to perform, and is inexpensive. For this reason, we think that it may represent an important analytical improvement. It has been suggested that nitrosothiols may exert biological activity by acting as a reservoir of NO. We tested the production of nitrite and of RSNO in stimulated, cultured murine microglial cells and we have shown that nitrite accumulates in these conditions. GSNO also accumulates, provided that GSH is present in the medium.