Effects of preadipocytes derived from mice fed with high fat diet on the angiogenic potential of endothelial cells.

BACKGROUND AND AIMS: Obesity promotes a persistent inflammatory process in the adipose tissue, activating the endothelium and leading to vascular dysfunction. Preadipocytes can interact with endothelial cells in a paracrine way stimulating angiogenesis. However, the potential of preadipocytes from adipose tissue of high fat diet (HFD) fed animal to stimulate angiogenesis has not been evaluated yet. The aim of this study was to investigate the effects of such diet on the angiogenic potential of preadipocytes in a mice model. METHODS AND RESULTS: We have evaluated body weight gain, fasting glucose levels and insulin resistance, mRNA expression in preadipocytes and endothelial cells after co-culture with preadipocytes, in vivo vascular function and in vitro endothelial cell migration and tubulogenesis. High fat diet promoted an increase in body weight, glycemic index and insulin resistance in mice. Preadipocytes mRNA expression of factors involved in angiogenesis was higher in these animals. In endothelial tEnd cells mRNA expression of factors involved in vessel growth were higher after co-culture with preadipocytes derived from mice fed with HFD. Although no significant differences were observed in in vivo vasodilatation response between control and HFD groups, endothelial tEnd cells showed an increase in migration and tubulogenesis when cultivated with conditioned media from preadipocytes derived from mice fed with HFD. CONCLUSION: Hypoxic and growth factors produced by preadipocytes derived from mice fed with HFD have higher capacity than preadipocytes derived from mice fed with standard diet to stimulate the angiogenic potential of endothelial cells, contributing to vascular disorders in obesity.

Microcirculatory effects of zinc on fructose-fed hamsters.

BACKGROUND AND AIMS: Fructose is a major dietary component directly related to vascular dysfunction and diseases such as obesity, diabetes, and hypertension. Zinc is considered a non-pharmacological alternative for treating diabetes due to its antioxidant and hyperglycemia-lowering effects in diabetic animals. Therefore, the aim of this study was to evaluate the effects of dietary zinc supplementation on the microcirculatory parameters of fructose-fed hamsters. METHODS AND RESULTS: Male hamsters (Mesocricetus auratus) were fed drinking water substituted by 10% fructose solution for 60 days, whereas control animals were fed drinking water alone. Their microcirculatory function was evaluated using cheek pouch preparation, as well as their blood glucose and serum insulin levels. Their microcirculatory responses to acetylcholine (ACh, an endothelium-dependent vasodilator) and to sodium nitroprusside (SNP, an endothelium-independent vasodilator) as well as the increase in macromolecular permeability induced by 30 min of ischemia/reperfusion (I/R) were noted. Endothelium-dependent vasodilation was significantly increased in control animals with high zinc supplementation compared to the groups without zinc supplementation. Zinc was able to protect against plasma leakage induced by I/R in all control and fructose-fed groups, although the microvascular permeability was higher in animals fed drinking water substituted by 10% fructose solution compared to those fed filtered drinking water alone. CONCLUSION: Our results indicate that dietary zinc supplementation can improve microvascular dysfunction by increasing endothelial-dependent dilatation and reducing the increase in macromolecular permeability induced by I/R in fructose-fed animals.

Effect of levofloxacin treatment on semen hyperviscosity in chronic bacterial prostatitis patients.

Changes in seminal fluid viscosity (SFV), reactive oxygen species (ROS) production, cytokines and seminal leucocyte concentration related to microbiological outcome in patients with chronic bacterial prostatitis (CBP) were studied. One hundred and ten infertile patients with CBP (positive sperm culture ≥10(5) colony-forming units [CFU] ml(-1), pathogens or Chlamydia in expressed prostatic secretions) were treated with levofloxacin 500 mg daily for 14 consecutive days per month for 3 months. In case of bacterial prostatitis, two conditions were examined: responders, eradication of 0 to <10(3) CFU ml(-1) (n = 78) and poor responders, >10(3) to <10(5) CFU ml(-1) (n = 32). Compared with poor responders, responders showed a significant increase of sperm progressive motility and a significant decrease in seminal leucocyte count, SFV, liquefaction time, ROS production (in all fractions and conditions), seminal tumour necrosis factor-α and interleukin 6. None of these variables showed significant differences compared with a control group of 37 fertile men. On the other hand, the poor responders showed significant changes in these variables compared with matched pretreatment values. In patients with CBP, antibiotic therapy alone leads to eradication in ≈71%, with improvement of sperm progressive motility, SFV and the framework of prooxidative factors. However, in the remaining ≈29% with poor antibiotic responsiveness, a deterioration of all variables is observed.

Relationship of semen hyperviscosity with IL-6, TNF-α, IL-10 and ROS production in seminal plasma of infertile patients with prostatitis and prostato-vesiculitis.

Changes in levels of oxidative damage products in semen and their relationship to seminal fluid viscosity (SFV) have recently received increasing research interest. We analysed whether SFV was associated with ROS generation, levels of cytokines TNF-alpha (TNF-α), IL-6 and IL-10 and seminal leucocyte concentration, and whether ROS production was related to the extent of infections/inflammations at one (prostatitis) or two (prostato-vesiculitis) male accessory glands. We studied 169 infertile patients, with chronic bacterial prostatitis (PR, n = 74) and/or bilateral prostato-vesiculitis (PV, n = 95), as diagnosed by the ultrasound (US) criteria. Healthy fertile men (n = 42) served as controls. In the PV patient group, SFV, semen characteristics and ROS production had median values that were significantly higher than those found in PR patients and controls, although other sperm variables had values significantly lower than those found in PR patients or controls. In PV infertile patients, ROS generation and pro-inflammatory cytokines levels were higher than those found in PR infertile patients and controls, although seminal IL-10 levels in PV and PR patients were lower than those found in the controls. In PR patients, the levels of SFV were positively related to TNF-α (r = 0.67; P < 0.01), fMLP-stimulated ROS production in the 45% Percoll fraction (r = 0.687, P < 0.01) and the 90% Percoll fraction in basal condition (r = 0.695, P < 0.01), and after fMLP-stimulation (r = 0.688, P < 0.01). Thus, our data indicated that seminal hyperviscosity is associated with increased oxidative stress in infertile men and increased pro-inflammatory interleukins in patients with male accessory gland infection, more when the infection was extended to the seminal vesicles.

Expression of STRBP mRNA in patients with cryptorchidism and Down's syndrome.

The most frequent defect of the male urogenital tract at birth is cryptorchidism. Cryptorchidism causes primitive testicular pathology responsible for infertility. Men with Down's syndrome (DS) have an increased risk of cryptorchidism. The spermatid perinuclear RNA-binding protein (STRBP) gene codifies a microtubule-associated RNA-binding protein and it is highly expressed in the testis as well as in the brain. At both levels, this gene seems to play a relevant role in the regular development of these organs. These observations prompted us to evaluate the expression of STRBP mRNA in 5 DS men with cryptorchidism and 5 normal healthy men (controls) by quantitative Real Time PCR in peripheral blood leukocytes. We found a decreased expression of the STRBP gene in men with DS and cryptorchidism compared with controls. This finding suggests that the impaired expression of this gene in DS may play a pathogenetic role in the altered brain and testicular development in subjects with DS and cryptorchidism.

Bone marrow-derived mononuclear cell therapy attenuates silica-induced lung fibrosis.

This study tests the hypothesis that bone marrow-derived mononuclear cell (BMDMC) therapy may reduce lung inflammation and fibrosis leading to an improvement in respiratory mechanics in a murine model of silicosis. 52 female C57BL/6 mice were randomly assigned into four groups. In the silica group (SIL), silica suspension (20 mg/50 µL in saline) was intratracheally instilled. In the control animals, 50 µL saline was administered intratracheally. At 1 h, the control and SIL groups were further randomised, receiving BMDMC (2×106 i.v. control-cell and SIL-cell) or saline (50 µL i.v. control and SIL). BMDMC were obtained from male donor mice. At day 15, lung mechanics, histology, and the presence of Y chromosome, interleukin (IL)-1ß, IL-1α, IL-1 receptor antagonist (IL-1RN), IL-1 receptor type 1, transforming growth factor (TGF)-ß and caspase-3 mRNA expressions in lung tissue were analysed. In the SIL-cell group, the fraction area of granuloma, the number of macrophages and the collagen fibre content were reduced, yielding improved lung mechanics. The presence of male donor cells in lung tissue was not confirmed using detection of Y chromosome DNA. Nevertheless, caspase-3, IL-1ß, IL-1α, IL-1RN and TGF-ß mRNA expression diminished after cell therapy. In conclusion, BMDMC acted on inflammatory and fibrogenic processes improving lung function through paracrine effects.

IL-6, TNFalfa, IL-10 in the seminal plasma of patients with bacterial male accessory gland infections after sequential therapy.

AIM: In previous studies the authors have demonstrated a worse spermatic outcome associated to overproduction of ROS in infertile patients with urogenital infections extended to more glands (prostato-vesciculitis, PV) compared to what observed in patients with prostatitis (P) (cat II according to the National Institutes of Health [NIH]). Among the reasons of an inadequate post-therapeutical response, the duration of each therapeutical phase could be the only bias of empirism entrusted only to the monitoring of obvious ''traditional'' end-points, resulting underrated for various reasons (costs, lacking methodological standardization, choice of the cytokines to be monitored). The evaluation of other therapeutical endpoints (cytokine dosage in the seminal plasma; analysis of ROS) is therefore all important. METHODS: In order to evaluate how to optimize the therapeutic response in infertile patients with P o PV chronic-bacterial, the authors wanted to monitor the pattern of the pro-oxidants cytokines TNFalfa, IL-6 in the seminal plasma (met. sandwich ELISA high sensitivity <0.039 pg/mL, R&D System Europe Ltd, UK) and of IL-10 (chosen as antioxidant cytokine) after sequential therapy (antibiotic - non-steroidal anti-inflammatory drugs antioxidant). RESULTS AND CONCLUSIONS: The modifications of the levels of TNFalfa, IL-6 ed IL-10 recorded in the present study during the sequential therapy for P or PV microbic offer some issues for reflection for interesting clinical-diagnostical implications: 1) possible revision of sequences and/or duration of the therapeutical phases in course of PV; and 2) the role to assign to the IL-10 (assumed as anti-inflammatory cytokine).

Synthetic peptides reproducing the EGF-receptor segment homologous to the pp60v-src phosphoacceptor site. Phosphorylation by tyrosine protein kinases.

The octapeptide E-E-K-E-Y-H-A-E, corresponding to the amino acid sequence 841-845 of EGF receptor, whose tyrosine-845 is homologous to the main phosphorylation site of pp60v-src, has been synthesized together with seven shorter peptides encompassing variable segments around the tyrosine residue. The peptides have been employed as model substrates for inspecting the local structural determinants of three tyrosine protein kinases (TPKs), namely; TPK-IIB and TPK-III, isolated from lymphoid cells (Eur. J. Biochem. 172, 451-457 (1988] and the TPK encoded by the oncogene of Abelson murine leukemia virus. The phosphorylation order with the different peptide substrates is variable depending on the TPK used: in particular, the lysine residue at position -2 relative to tyrosine proved especially harmful with TPK-IIB, the peptides K-E-Y-H and K-E-Y-H-A-E being very poor substrates compared with their shorter derivatives devoid of the N-terminal lysine (E-Y-H and E-Y-H-A-E, respectively). Conversely, such a basic residue is well tolerated by the other two TPKs. The negative effect of the N-terminal lysine on TPK-IIB-catalyzed phosphorylation is accounted for by an increase of Km and can be overcome by the presence of additional glutamic acid(s) on that side. On the other hand, the C-terminal acidic doublet Ala-Glu specifically impairs the phosphorylation efficiency of abl-TPK, by lowering the Vmax value, the heptapeptide E-K-E-Y-H-A-E being much less readily phosphorylated than E-K-E-Y-H. Collectively, these results would indicate that the site specificity of tyrosine protein kinases results from the balance of positive and negative determinants whose influence on the catalytic activity of the individual enzymes can differ greatly.

[Unilateral testicular cancer: incidence and effects of a contralateral testiculopathy on the sperm output]. / Neoplasia testicolare monolaterale: frequenza ed effetti di una coincidentale testiculopatia controlaterale sul quadro spermatico.

AIM: The aim of this paper was to evaluate the incidence of a non-tumoral, contralateral primitive testiculopathy and its relative influence on sperm quality of patients with unilateral testicular cancer. METHODS: Twenty-four patients (mean age 26 years, range 19-38) with testicular germ cell cancer (seminomas, SEM, in 10 cases; nonseminomas, NSEM, in the remaining 14 patients) after orchiectomy and before radiotherapy or chemotherapy underwent semen analysis, physical examination and scrotal ultrasound of their survivor testis. RESULTS: Patients with SEM had sperm concentration, total sperm count and forward motility significantly higher than those found in patients with NSEM. Altogether, 5 out of 24 patients (2 SEM; 3 NSEM) (20.8%) showed azoospermia; 10 patients (41.7%) (3 SEM; 7 NSEM) had oligo-, astheno- and/or terato-zoospermia (OAT). The remaining 9 patients (37.5%) (5 SEM; 4 NSEM) showed normal sperm parameters. The testicular volume of the left over testis was reduced (<12 ml) in 4 out of 5 (80%) azoospermic patients, in 7 out of 10 patients (70%) of OAT patients, but in no patient (0%) with normozoospermia. A testicular biopsy performed on the survivor testis of 5 patients with azoospermia (4 of them had a reduced testicular volume) confirmed the primitive testiculopathy, showing a histological pattern of Sertoli cell syndrome only in 4 of them (80%) and maturation arrest in the other case (20%). CONCLUSIONS: Less (OAT) or more severe (azoospermia) sperm output impairment in patients with unilateral testicular cancer is associated with a coincidental, contralateral to unilateral testicular cancer, primitive testiculopathy expressed as reduced testicular volume and impairment spermatogenesis at the testicular biopsy.

Effect of substance P and its natural analogues on gastric emptying of the conscious rat.

1 Substance P and its natural analogues were tested for their effect on gastric emptying in the rat. 2 Substance P and kassinin were virtually inactive even at the maximum dose tests. 3 The other tachykinins significantly delayed gastric emptying their effect being quite remarkable with 100 micrograms/kg. The lowest doses (30 microgram/kg) caused a slight and non-significant increase in emptying rate. 4 The effect on gastric emptying was most probably correlated with a spasmogenic effect on the gastroduodenal junction, as pointed out in previous work.

Relative potency of bombesin-like peptides.

The pharmacological activity of two natural bombesin-like peptides, alytesin and litorin, and 25 related synthetic peptides has been compared to that of bombesin. 2 The minimum length of the amino acid chain required for the first appearance of bombesin-like effects was represented by the C-terminal heptapeptide, and the minimum length for maximal effects by the C-terminal nonapeptide. The latter possessed approximately the same activity as bombesin and may be considered a good substitute. 3 Both the tryptophan and histidine residues seemed to be essential for bombesin-like activity. 4 The C-terminal octapeptide was less active than either bombesin or the C-terminal nonapeptide and its action was more rapid in onset and less sustained. 5 Litorin apparently has an intermediate position between bombesin octapeptide and bombesin nonapeptide in the speed and duration of its effects. The relationship between structure and activity is discussed.

Structural requirements of ceruletide-like peptides for activity on gut and brain.

A series of ceruletide analogues were tested in order to evaluate the structural requirements for activity on gut and brain. The effects studied were: contraction of the guinea pig gallbladder and stimulation of the exocrine pancreatic secretion in the dog after intravenous (IV) administration, for activity on the gut; analgesia (hot plate test) and sedation (reduction of spontaneous rearing activity) in mice by subcutaneous (SC) route, for activity on the brain. The structural requirements were roughly the same for central and peripheral activities. Possible mechanisms for the antinociceptive effects of ceruletide in humans were proposed.

Structure-activity relationships of dermorphin synthetic analogues.

A number of dermorphin analogues have been reviewed for antinociceptive activity after systemic administration. Analgesic peptides as potent as or more potent than dermorphin are found among the hepta- and tetrapeptide analogues, probably because of enhanced stability towards carboxyldipeptidases imparted by the Pro6 and D-Ala2 residues lying on the right and left side, respectively, of the point of cleavage. Hexapeptide analogues are practically inactive. [D-Arg2]derivatives are very potent, particularly in the tetrapeptide series. In binding studies dermorphin behaves as a pure opioid agonist, with a marked affinity and selectivity for the mu-type opioid receptors. In the heptapeptide analogues a direct correlation seems to exist between mu-selectivity and "sodium shift," and between lipophilicity and MVD/GPI potency ratio.

Opioid receptor binding profile of selected dermorphin-like peptides.

The receptor binding profile of a selected group of dermorphin-like peptides was determined and correlated with the results of the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and with the currently used antinociception tests in the rat. For the peptides with the characteristic dermorphin D-Ala2-Phe3-Gly4 sequence, a linear negative correlation was found between the reciprocal of sodium shift and relative affinity for the mu-type opioid receptor. For the same peptides, a positive correlation was evidenced between relative potency on GPI and MVD and relative affinity for mu- and delta-type receptors, respectively.

Stimulatory action of (beta-Asp9) ceruletide on dog pancreatic exocrine secretion.

The synthetic analog of ceruletide, (beta-Asp9) ceruletide, which in previous studies was found to retain some central effects of ceruletide with virtually no effect on gallbladder contraction, was studied for its stimulatory effect on pancreatic secretion. This peptide was shown to induce an increase in the volume of pancreatic juice similar to that provoked by the parent compound. Its activity was 4% of that of ceruletide, as regards volume, dry residue and protein content of the pancreatic juice. It was concluded that the dissociation between central and peripheral effects concerned only the gallbladder contractility, since its potency as a pancreatic stimulant was of the same order of magnitude as that shown on the central nervous system.

Antinociceptive and other opioid effects of a new series of dermorphin analogues after subcutaneous administration in the rat.

A series of hepta-, hexa-, penta- and tetrapeptide analogues of dermorphin have been evaluated in the rat for antinociceptive activity after subcutaneous (SC) administration at the screening dose of 4 mg/kg. Effective doses (ED50) were calculated for the most active compounds. Presence of spontaneous movements, defecation, micturition and corneal reflex were also recorded. Syntheses and analytical data of new derivatives are briefly reported.

Phyllomedusa skin: a huge factory and store-house of a variety of active peptides.

The skin of the neotropical hylid frogs belonging to the subfamily. Phyllomedusinae is a formidable factory and store-house of a variety of active peptides belonging to seven distinct families: the caeruleins (represented by phyllocaerulein), the bradykinins (phyllokinin), the tachykinins (phyllomedusin), the bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdei-litorin), sauvagine, the dermorphins (dermorphin, [Hyp6]dermorphin) and finally the tryptophyllins (a set of 8-11 members). Another linear peptide and three diketopiperazines should be added to the list. The biochemical and pharmacological positions of the Phyllomedusa peptides within their families is briefly discussed, dwelling upon some recent and controversial data.

Antinociceptive, prolactin releasing and intestinal motility inhibiting activities of dermorphin and analogues after subcutaneous administration in the rat.

A series of analogues and shorter homologues of dermorphin (DM), a frog skin heptapeptide with potent morphine-like activity, have been assayed in the rat after subcutaneous (SC) administration at the screening dose of 4 mg/kg. The effects taken into account are: analgesia (tail-pinch test), stimulation of prolactin (PRL) secretion, and inhibition of gastro-intestinal (GI) motility (charcoal meal transit). Effective doses were calculated for the most active compounds. The potency of DM (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) in the different tests was: tail-pinch: ED50 = 0.83 mg/kg; PRL release: ED100 = 0.3 mg/kg; inhibition of GI motility: ED30 = 1.8 mg/kg.

Alanine scan of endothelin: importance of aromatic residues.

A systematic approach to map the functional important determinants of endothelin-1 (ET) by an alanine scan is described. Studies on the in vitro receptor binding affinity and on the agonist contracting activity defined that residues Asp8, Tyr13, Phe14, Leu17, and Trp21 were of major biological significance. A striking observation was that four out of these five sites were hydrophobic amino acids. Ala analogues of the aromatic residues at position 13, 14, and 21 displayed sharply reduced receptor binding affinity (< 2% of ET) and can be considered important for receptor contact. Ala analogues of Asp8 and Leu17 lost most (> 90%) of the agonist activity but retained a receptor affinity nearly equivalent to ET and can be considered to be important for signal transduction. Three other positions, Val12, Asp18, and Ile20 (which are adjacent to the biologically important sites of Tyr13, Leu17, and Trp21), resulted as partially tolerant to Ala substitution, retaining 14-50% of the potency of ET. Ala analogues of the Et isomeric disulfide arrangement (Cys1,11 and Cys3,15) were always less active than the corresponding analogues with the native disulfide pairings (Cys1,15 and Cys3,11).

Synthetic peptides related to the dermorphins. I. Synthesis and biological activities of the shorter homologues and of analogues of the heptapeptides.

Dermorphins are potent opiate-like peptides isolated from the skin of some species of frogs. They are characterized by the presence of a D-amino acid residue, which is crucial for bioactivity. A number of analogues were prepared in order to evaluate the structure-activity relationships. The syntheses were accomplished either by conventional or solid-phase procedures. In vitro assays included both guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Central analgesic (tail-flick and hot plate tests) and cataleptic activities were determined in the rat by intracerebroventricular route. The potency of dermorphin (H- Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) in the different tests was: GPI: IC50 = 3.3 nM; MVD: IC50 = 29 nM; hot plate: ED50 = 13.3 pmol/rat; tail-flick: ED50 = 23 pmol/rat; catalepsy: ED50 = 130 pmol/rat.