Understanding the role of haptoglobin in psoriasis: effects of ultraviolet B.

BACKGROUND: Haptoglobin (Hp) is one of the acute phase proteins, whose main function is to bind free haemoglobin (Hb) and transport it to the liver for degradation and iron recycling. In addition to its role as an Hb scavenger, Hp has been shown to behave as an anti-inflammatory, antioxidant and angiogenic factor. We previously investigated the role of Hp in the pathogenesis of psoriasis, and found that it displays some structural modifications that might be associated with protein function in the disease. Phototherapy is an efficacious treatment for psoriasis, although the biological mechanisms by which phototherapy improves psoriasis are still unclear. AIM: To investigate the effects of ultraviolet (UV)B on Hp to clarify the role of Hp in psoriasis. METHODS: Expression of the genes encoding Hp, interleukin (IL)-6 and IL-10 was assessed in UVB-irradiated and unirradiated HaCaT cells. The biological significance of Hp modulation of UVB treatment was confirmed by ELISA and Western blotting. The Hp gene and protein expression in the skin of patients with psoriasis was also investigated. RESULTS: In vitro results showed that UVB modulated IL-6 and IL-10 gene expression and Hp gene and protein expression in HaCaT cells. The in vivo data also showed that Hp levels were increased in the skin of patients with psoriasis compared with healthy controls. CONCLUSIONS: UVB irradiation was able to modulate Hp production in immortalized keratinocytes. The higher levels of Hp in vivo in both lesional and nonlesional skin suggest that it might have a role in the pathogenesis of the disease.

Transglutaminase-mediated modifications of the rat sperm surface in vitro.

Two transglutaminase-mediated modifications of the rat epididymal spermatozoon surface were demonstrated in vitro. Transglutaminase was effective in promoting the binding of spermidine to the sperm. Moreover, the enzyme, by reacting with one of the major proteins secreted by the rat seminal vesicle epithelium, produced a modified form of the protein with a higher molecular weight and the capability of binding to the sperm cells. A specific physiological role for the enzyme, bringing about modifications of the rat sperm surface in the seminal fluid environment, is suggested.

Haptoglobin from psoriatic patients exhibits decreased activity in binding haemoglobin and inhibiting lecithin-cholesterol acyltransferase activity.

OBJECTIVE: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of haptoglobin (Hpt). BACKGROUND: Hpt is a plasma acute-phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free haemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT). STUDY DESIGN: Hpt was isolated from patients with psoriasis vulgaris, and its activity in haemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. METHODS: Two affinity chromatography steps, the first using resin-coupled haemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by Enzyme-linked immunosorbent assay with stationary haemoglobin or ApoA-I, Hpt in solution and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. RESULTS: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by haemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both haemoglobin binding and LCAT inhibition. CONCLUSIONS: In psoriasis, Hpt displays some structure modification(s), which might be associated with the protein function in the disease.

Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis.

Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pd Delta) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pd Delta and wild-type (wt) ES cells. We found that G6pd Delta ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-X(L). Bcl-X(L) does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Interestingly, ERK activation has opposite outcomes in G6pd Delta ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level.

Detection of sperm-coating antigens immunologically related to a seminal protein in rat.

We report in this paper that proteins from the surface of ejaculated spermatozoa contain antigenic determinants cross-reacting with a rabbit antiserum raised against native CFS, a protein secreted from the rat seminal vesicle and composed of two subunits, namely RSV IV and RSV V. Conversely, no such proteins could be extracted from cauda epididymal spermatozoa. The cross-reacting proteins derived from the ejaculated spermatozoa were analyzed by SDS-PAGE. An electrophoretic pattern different than that expected for native CFS in denaturing conditions was found. In vitro reconstitution experiments showed that labeled native CFS is able to bind cauda epididymal spermatozoa. The CFS protein recovered from the sperm surface was examined and alterations of its structure were also noted. The sperm-coating abilities of CFS and of its RSV IV subunit are discussed.

CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface.

A soluble 1, kDa glycoprotein, namely gp17, was previously isolated from human semen and used to obtain mouse monoclonal or chicken polyclonal antibodies. This protein was shown to bind CD4+ T-cells and to soluble recombinant CD4 in vitro. Here, we report that the anti-gp17 monoclonal antibodies are captured by ejaculated spermatozoa and that gp17-like antigens are released by cell acid extraction. Immunoblotting experiments with monoclonal antibodies indicated that SDS-lysates from spermatozoa contain proteins with the same electrophoretic and antigenic properties of CD4 and gp17. Anti-CD4 mouse monoclonal antibodies were used to coprecipitate from NP40-lysate proteins reacting with chicken anti-gp17 antibodies. Analytical chromatography demonstrated that a number of gp17-like forms are present in the seminal plasma, put that only the 1 kDa species can be detected in the spermatozoa lysate. This protein was localised by immunofluorescence on the post-acrosomal region of the spermatozoon. The same surface domain was also reactive with anti-CD4 antibodies. After treatment to induce in vitro capacitation, gp17 was detected all over the spermatozoon head. Conversely, only a minor part of the treated spermatozoa exhibited CD4 immunostaining, which remained localised on the post-acrosomal region. The possible function of CD4 and gp17 on male germ cells is discussed.

Synthesis of ascorbate and urate in the ovary of water buffalo.

Blood flow interruption is associated with oxygen depletion and loss of factors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We studied the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were aspirated from each antral follicle, before and during incubation of the ovaries at 39 degrees C. Urate, ascorbate, retinol and alpha-tocopherol in the fluid were, titrated by High Performance Liquid Chromatography (HPLC) with spectrophotometric or spectrofluorimetric detection. The total antioxidant capacity of follicular fluid was determined as absorbance decrease, following addition of a source of radical chromophores. The more the incubation progressed, the higher levels of urate, ascorbate and total antioxidant capacity were found. Conversely, changes in concentration of the liposoluble antioxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-gamma-lactone oxidase in microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD-p-nitroanilide cleaving activity, was found related with the immunoreactivity to anti-CPP32 antibodies. Negative correlation between the enzyme activity (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of nitrotyrosine, a product of peroxynitrite attack on proteins, was measured in follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amount was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concentrations of ascorbate or urate may be associated with oxidative stress during follicular atresia.

Relative ratios of lactoferrin, albumin, and acid phosphatase seminal levels as sperm quality markers in fertile and infertile men.

Human seminal plasma proteins from fertile and infertile men were fractionated by electrophoresis. The amounts of three Coomassie-stained protein bands were measured by densitometry. Their relative ratios were constant in normospermic men but varied in the infertile patients. Laboratory manipulation of the semen was shown not to affect the protein ratios as observed after liquefaction, incubation at various temperatures, and storage at -20 degrees C. The three proteins were purified by chromatographic techniques and identified as lactoferrin, albumin, and acid phosphatase by electrophoresis, high-pressure liquid chromatography, and enzyme assays. The use of these proteins to evaluate the contributions of different fluids to seminal plasma is discussed.

Evaluation of the antioxidant response in the plasma of healthy or hypertensive subjects after short-term exercise.

Reactive oxygen species are produced during exercise. The antioxidants prevent or limit tissue damages by these species in physiological conditions. In particular, ascorbate and urate scavenge peroxynitrite, which can alter the function of many molecules, including the lecithin-cholesterol acyltransferase (LCAT) enzyme involved in reverse cholesterol transport. The aims of the present study were to compare the plasma antioxidant response to an ergometric test (ET) in hypertensive and healthy subjects, evaluate the exercise-dependent nitrosative stress in plasma, and assess whether the LCAT activity is altered by the exercise. Plasma samples, prepared before and after ET from hypertensive or healthy volunteers, were analysed for their levels of ascorbate, urate, alpha-tocopherol, retinol, nitrotyrosine, and LCAT activity. The alpha-tocopherol and retinol levels did not significantly change in both groups during exercise, while the ascorbate level changed displaying higher increase in controls (+38.8%) than in hypertensives (+17.2%). In these patients, during ET, the urate and nitrotyrosine levels changed more than in normotensives (+13.5 and +40.6% vs -3.1 and +25.2%, respectively). The antioxidants effectively prevented loss or reduction of LCAT activity, as it was similar in hypertensives and normotensives, and did not change after ET. The results demonstrate that exercise is associated with enhanced protein nitrosation, and suggest that the ascorbate or urate levels increase to limit oxidative damage.

Estradiol esterification in the human preovulatory follicle.

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K(m) and V(max) values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.

Lecithin-cholesterol acyltransferase activity during maturation of human preovulatory follicles with different concentrations of ascorbate, alpha-tocopherol and nitrotyrosine.

The enzyme lecithin-cholesterol acyltransferase (LCAT) transfers an acyl chain from lecithin to cholesterol or oestradiol, thus playing a crucial role in reverse cholesterol transport and follicular synthesis of potent long-lived oestrogens. The mechanism of catalysis is biphasic, as it is based on a phospholipase and an esterifying activity. Sulfhydryl groups were previously reported to be required for the esterification step. Lecithin-cholesterol acyltransferase has previously been shown to be inhibited by thiol oxidants such as peroxynitrite. Peroxynitrite also converts tyrosine to nitrotyrosines. In the present study, high levels of nitrotyrosine associated with low LCAT activity, and vice versa, were found in human preovulatory follicular fluids. Follicular fluids were also analysed for oestradiol (E) and progesterone (P) concentrations. The E/P ratio, which decreases as ovulation approaches, was used to evaluate the maturation status of each follicle. Enzyme activity was negatively correlated with the E/P ratio. Ascorbate (Asc) and alpha-tocopherol (Toc) were titrated in follicular fluid and plasma to evaluate their accumulation or consumption in the follicle. High LCAT activity was found in follicular fluids where Asc and Toc had accumulated, whereas lower activity was associated with Asc and Toc consumption. The consumption of both antioxidants was positively correlated with the E/P ratio. The results suggest that as follicle maturation progresses, Toc and Asc concentrations increase in follicular fluid, thus protecting LCAT from oxidative damage and loss of activity.

The accumulation of alpha-Tocopherol and Retinol in the milk of water buffalo is correlated with the plasma levels of triiodothyronine.

Milk is the most important source of Retinol and alpha-Tocopherol for calves. These antioxidants save the food quality and prevent lipid oxidation in the mammary gland and the calf growing tissues. In Bubalus bubalis, seasonal changes for the plasma levels of both antioxidants were not found. The levels of Retinol and alpha-Tocopherol in the milk were 2 and 1.7 times higher in winter than in summer, respectively. These levels were correlated with the plasma level of triiodothyronine, and markedly increased in cows injected with triiodothyronine in summer. The cytosol from alveolar epithelial cells of mammary glands was incubated with alpha-Tocopherol and 3H-Retinol and, after gel filtration chromatography, both antioxidants were found associated with proteins migrating as a single peak of 33 kD. The amount of alpha-Tocopherol and Retinol binding proteins was 1.5 and 2.3 times higher in winter than in summer respectively. The Retinol binding proteins migrated as two bands (33 and 16 kD) by electrophoresis in denaturing and reducing conditions. Our data suggest that triiodothyronine enhances the transport of both liposoluble antioxidants through the blood-mammary barrier, and demonstrate that proteins of the mammary epithelial cells are involved in such a transport.

Zinc-protein from rat prostate fluid binds epididymal spermatozoa.

The detection and the isolation of a zinc-protein from the secretion of the rat dorsolateral prostate is described. The purification procedure, based on gel filtration and cationic exchange chromatography, allowed to separate a minor protein (Mr approximately 66,000) from free zinc ions and other secretory components. Two zinc ions were estimated to be associated with one molecule of isolated protein. The zinc-protein was labelled with 125I and then incubated at 37 degrees C with spermatozoa from rat epididymal cauda. Time-dependent in vitro binding of the radioactive protein to sperm cells was demonstrated. This binding was not affected by the presence of proteins from the seminal vesicle during the incubation, while it was blocked in the presence of an excess of unlabelled zinc-protein. After binding, the labelled spermatozoa were treated with a buffer containing 0.5% sodium deoxycholate and 40 mM EDTA; only very small amounts of label were removed from the cells, thus suggesting that the zinc-proteins were kept on the plasma membrane by interactions which do not involve merely hydrophobic bonds.

A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa.

The rat seminal vesicle produces large amounts of a protein-rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm-coating antigen. We report in this paper that high-molecular-weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm-bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high-molecular-weight, sperm-coating antigen.

Binding of free and protein-associated zinc to rat spermatozoa.

1. The zinc content of rat spermatozoa increases, upon ejaculation, from 0.035 to 1.055 micrograms/10(6) cells. 2. The rat seminal plasma holds zinc both as free ion and as protein-bound forms. 3. Zinc-free ions bind in vitro to rat epididymal spermatozoa. 4. Zinc-protein complexes can be isolated, by a chromatographic procedure, from the dorsolateral lobe of rat prostate. 5. The isolated zinc-protein complexes bind in vitro to rat epididymal spermatozoa.

Interaction of seminal plasma proteins with cell surface antigens: presence of a CD4-binding glycoprotein in human seminal plasma.

We report in this paper the presence in the human seminal plasma of a glycoprotein capable of binding to CD4, a surface antigen expressed on the surface of T-cells, macrophages, and sperm cells, which acts as a coreceptor in antigen-mediated T-cell activation and as a receptor for the AIDS virus, HIV-1. This protein, namely gp17 (apparent MW = 17,500 Da), was purified by affinity chromatography and characterized by SDS/PAGE analysis. Its binding to CD4 was inhibited by anti-CD4 mAbs directed against V1, a region of CD4 implicated in the binding to MHC class II antigens and to the HIV-1 envelope protein gp120, but not by mAbs directed against other CD4 determinants. The presence of a CD4-masking factor in human seminal plasma may be relevant to the modulation of maternal immunity at insemination and to the control of sexual transmission of HIV-1.

Interaction of proteins RSV IV and RSV V in rat seminal vesicle secretion.

The RSV IV polypeptide, molecular weight ratio (Mr = 10,000), which is produced by the rat seminal vesicle, has previously been suggested to be associated with another polypeptide in the gland secretion (Higgins et al., '76). This study provides that RSV IV is a component of a protein shown by immunoassays, electrophoresis, and amino acid composition analysis to contain, together with RSV IV, the seminal vesicle secretory RSV V polypeptide (Mr = 13,000). This RSV IV-RSV V complex (namely CFS protein) had an isoelectric point at pH 7.2 and an approximate molecular weight of 22,000 daltons. This complex inhibits the previously reported in vitro binding of the isolated RSV IV to epididymal sperm cells, thus suggesting a functional role for the RSV IV-RSV V interaction.

The 11S rat seminal vesicle mRNA directs the in vitro synthesis of two precursors of the major secretory protein IV.

The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.