Glutamate-induced calcium increase in myotubes depends on up-regulation of a sodium-dependent transporter

We report a study on the regulation by 2-chloro adenosine (2CA) of a glutamate (Glu) transporter in myogenic C2C12 cells. Long-term 2CA exposition significantly increased the V(max) of the Glu transporter. Moreover, 2CA-treated cells responded to Glu challenge by a rapid and transient increase in their intracellular calcium level. The above reported effects were totally abolished by treating C2C12 cells with the Na(+)-dependent Glu transporter inhibitors DL-threo-b-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid. We propose that the possible link between the Glu uptake increase and the Glu induction of calcium rise could be the depolarizing currents carried by Na(+) coupled with transporter activity.     

Fine structure of leydig and sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata

An ultrastructural investigation revealed the presence of true Leydig cells in the testis of sexually mature specimens of Torpedo marmorata. They showed the typical organization of steroid-hormone-producing cells, which, however, changed as spermatocysts approached maturity. In fact, they appeared as active cells among spermatocysts engaged in spermatogenesis, while in regions where spermiation occurred, they progressively regressed resuming the fibroblastic organization typically present in the testis of immature specimens. Such observations strongly suggest that these cells might be engaged in steroidogenesis and actively control spermatogenesis. Sertoli cells, too, appeared to play a role in spermatogenesis control, since, like Leydig cells, they showed the typical aspect of steroidogenic cells. In addition, the presence of gap junctions between Sertoli cells suggests that their activity might be coordinated. After sperm release, most Sertoli cells were modified and, finally, degenerated, but few of them changed into round cells (cytoplasts) or round cell remnants, which continued their steroidogenic activity within the spermatocyst and the genital duct lumen. From the present observations, it can be reasonably concluded that, in T. marmorata, spermatogenesis depends on both Leydig and Sertoli cells, and, as postulated by Callard (1991), in cartilaginous fish, the function of the Leydig cells as producers of steroids might be more recent and subsequent to that of Sertoli cells. In this regard, it is noteworthy that, in immature males, when Leydig cells showed a fibroblastic organization, Sertoli cells already displayed the typical organization of a steroidogenic cell.     

Retinol-binding protein-deficient mice: biochemical basis for impaired vision

We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice.     

Effects of docosahexaenoic acid on vascular pathology and reactivity in hypertension

Previous studies have shown that docosahexaenoic acid (DHA) has an antihypertensive effect in spontaneously hypertensive rats (SHR). To investigate possible mechanisms for this effect, vascular pathology and reactivity were determined in SHR treated with dietary DHA. SHR (7 weeks) were fed a purified diet with either a combination of corn/soybean oils or a DHA-enriched oil for 6 weeks. Histological evaluation of heart tissue, aorta, coronary, and renal arteries was performed. Vascular responses were determined in isolated aortic rings. Contractile responses to agonists, including norepinephrine (10(-9) to 10(-4) M), potassium chloride (5-55 mM), and angiotensin II (5 x 10(-7) M) were assessed. Vasorelaxant responses to acetylcholine (10(-9) to 10 (-4) M), sodium nitroprusside (10(-9) to 10(-6) M), papaverine (10(-5) to 10(-4) M), and methoxyverapamil (D600, 1-100 microM) were determined. DHA-fed SHR had significantly reduced blood pressure (P < 0.001) and vascular wall thicknesses in the coronary, thoracic, and abdominal aorta compared with controls (P < 0.05) Contractile responses to agonists mediated by receptor stimulation and potassium depolarization were not altered in DHA-fed SHR. Endothelial-dependent relaxations to acetylcholine were not altered which suggests endothelial-derived nitric oxide production/release is not affected by dietary DHA. Other mechanisms of vascular relaxation, including intracellular cyclic nucleotides, cGMP, and cAMP were not altered by dietary DHA because aortic relaxant responses to sodium nitroprusside and papaverine were similar in control and DHA-fed SHR. No significant differences were seen in relaxant responses to the calcium channel blocker, D600, or contractile responses to norepinephrine in the absence of extracellular calcium. These results suggest that dietary DHA does not affect mechanisms related to extracellular calcium channels or intracellular calcium mobilization. Moreover, the contractile and vasorelaxant responses are not differentially altered with dietary DHA in this in vivo SHR model. The findings demonstrate that dietary DHA reduces systolic blood pressure and vascular wall thickness in SHR. This may contribute to decrease arterial stiffness and pulse pressure, in addition to the antihypertensive properties of DHA. The antihypertensive properties of DHA are not related to alterations in vascular responses.     

The impact of different antioxidant agents alone or in combination on reactive oxygen species,antioxidant enzymes and cytokines in a series of advanced cancer patients at different sites: correlation with disease progression

In the present study we tested the ability of different antioxidant agents, used alone or in combination, to reduce the reactive oxygen species (ROS) levels and to increase the glutathione peroxidase (GPx) activity. Moreover, we tested the ability of such antioxidant agents to reduce the serum levels of proinflammatory cytokines IL-6 and TNFalpha. Fifty-six advanced stage cancer patients with tumors at different sites were included in the study: they were mainly stage III (12.5%) and stage IV (82.1%). The study was divided into two phases. In the 1st phase 28 patients were divided into five groups and a single different antioxidant agent was administered to each group. The selected antioxidant agents were: alpha lipoic acid or carboxycysteine-lysine salt, amifostine, reduced glutathione, vitamin A plus vitamin E plus Vitamin C. In the 2nd phase of the study 28 patients were divided into five groups and a combination of two different antioxidant agents was administered to each group. The antioxidant treatment was administered for 10 consecutive days. The patients were studied at baseline and after antioxidant treatment. Our results show that all single antioxidants tested were effective in reducing the ROS levels and three of them in increasing GPx activity, too. Among the combinations of antioxidant agents, three were effective in reducing ROS, while three were effective in increasing GPx activity (arm 4 was effective in both instances). Comprehensively, the "antioxidant treatment" was found to be effective both on ROS levels and GPx activity. Moreover, the antioxidant treatment was able to reduce serum levels of IL-6 and TNFalpha. Furthermore, a correlation was shown between the Eastern Cooperative Oncology Group Performance Status of patients and blood levels of ROS, GPx activity, serum levels of proinflammatory cytokines.     

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.     

Rescue of Fructose-Induced Metabolic Syndrome by Antibiotics or Faecal Transplantation in a Rat Model of Obesity

A fructose-rich diet can induce metabolic syndrome, a combination of health disorders that increases the risk of diabetes and cardiovascular diseases. Diet is also known to alter the microbial composition of the gut, although it is not clear whether such alteration contributes to the development of metabolic syndrome. The aim of this work was to assess the possible link between the gut microbiota and the development of diet-induced metabolic syndrome in a rat model of obesity. Rats were fed either a standard or high-fructose diet. Groups of fructose-fed rats were treated with either antibiotics or faecal samples from control rats by oral gavage. Body composition, plasma metabolic parameters and markers of tissue oxidative stress were measured in all groups. A 16S DNA-sequencing approach was used to evaluate the bacterial composition of the gut of animals under different diets. The fructose-rich diet induced markers of metabolic syndrome, inflammation and oxidative stress, that were all significantly reduced when the animals were treated with antibiotic or faecal samples. The number of members of two bacterial genera, Coprococcus and Ruminococcus, was increased by the fructose-rich diet and reduced by both antibiotic and faecal treatments, pointing to a correlation between their abundance and the development of the metabolic syndrome. Our data indicate that in rats fed a fructose-rich diet the development of metabolic syndrome is directly correlated with variations of the gut content of specific bacterial taxa.     

Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.