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G Forti

Publications and source records attributed to G Forti.

At least 37 records · Page 2Linked to original sources
PubMed2000

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.

Acetyltransferases↗
PubMed2000

Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction.

Two processes, namely capacitation and acrosome reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility properties of spermatozoa render these cells able to penetrate oocyte investments and responsive to stimuli that induce acrosome reaction prior to fertilization. Physiological acrosome reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and expose molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and acrosome reaction have been partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodeling in sperm plasma membrane as well as changes in protein phosphorylation. Some of the kinases and phosphorylated proteins that are involved in the processes of capacitation and acrosome reaction have been now characterized. Characterization of sperm receptors to physiological inducers of acrosome reaction is in progress. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.Furthermore, the mechanisms underlying sperm DNA fragmentation are also briefly reviewed.

Acrosome Reaction↗
PubMed2000

A novel functional estrogen receptor on human sperm membrane interferes with progesterone effects.

We have identified an estrogen receptor of approximately 29 kDa apparent molecular weight in human sperm membranes by ligand and Western blot analysis, respectively, using peroxidase-conjugated E(2) and an antibody directed against the ligand binding region of the genomic receptor (alphaH222). Such receptor is functional since 17betaE(2) induces a rapid and sustained increase of intracellular calcium concentrations ([Ca(2+)](i)) which is completely eliminated by preincubation with alphaH222. 17betaE(2) effects on calcium are clearly mediated by a membrane receptor, as they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E(2). Dose-response curve for this effect is biphasic with EC(50)s in the nanomolar and micromolar range. In addition to calcium increase, 17betaE(2) stimulates tyrosine phosphorylation of several sperm proteins including the 29-kDa protein band. Preincubation of human sperm with 17betaE(2) inhibits calcium and acrosome reaction increases in response to progesterone. We conclude that estrogens may play a role in the modulation of non-genomic action of progesterone (P) in human sperm during the process of fertilization.

Acrosome Reaction↗
PubMed2000

Duplex ultrasound evaluation of cavernosal peak systolic velocity and waveform acceleration in the penile flaccid state: clinical significance in the assessment of the arterial supply in patients with erectile dysfunction.

The aim of this paper was to establish if duplex ultrasound parameters obtained for assessment of the patency of cavernosal arteries in the penile flaccid state can give sufficient clinical information without the use of intracavernosal injection of vasodilatory drugs. We assessed mean cavernosal peak systolic velocity (PSV) in the penile flaccid state (basal PSV), and after PGE1 injection (dynamic PSV) in 339 unselected patients with erectile dysfunction. In 55 of these patients the waveform acceleration in the flaccid state was also assessed. The results of the study can be summarized as follows: (1) a significant relationship was found between basal and dynamic PSV in the 339 patients (r=0.477; p < 0.0001); (2) a basal PSV >12.5 cm/sec was predictive of a dynamic PSV >/=30 cm/sec in 129/139 (92.8%) of the patients, whereas in patients with a basal PSV </=12.5 a dynamic PSV both > or <30 cm/sec could be found; and (3) an acceleration >1 m/sec2 in the flaccid state was coupled to a dynamic PSV >30 cm/sec in 43/46 (93.5%) of the patients independent of the basal PSV. In conclusion, these results suggest that the combined duplex ultrasound assessment of PSV and waveform acceleration in the penile flaccid state can predict arterial dynamic inflow in the majority (51/55; 92.7%) of patients with erectile dysfunction, with less time and expense and less discomfort for the patient.

Alprostadil↗
PubMed2000

Functional adenosine receptors in human corpora cavernosa.

We have demonstrated that adenosine has potent relaxant activity on the rabbit corpus cavernosum, acting through the A2a subtype receptor for adenosine. We now report studies on the identification and functional characterization of adenosine receptors in human penile vessels. To identify A2 receptors in human corpora cavernosa (HCC) we performed binding studies using the selective radioligand [125I]PAPA-APEC in membranes from HCC. We found the presence of a single class of high affinity (Kd= 0.23 +/- 0.06 nM), low capacity (Bmax=134 +/- 37 fmoles/mg protein) binding sites. Adenosine and CGS 21680 completely displaced [125I]PAPA-APEC binding (Kd= 146.7 +/- 64 microM and 51.52 +/- 27 nM, respectively). Accordingly, in functional studies adenosine relaxed phenylephrine precontracted HCC with an IC50=2.28 +/- 0.17 mM. The effect of adenosine was independent from nitric oxide (NO), and was counteracted by the A2 antagonist CGS 15943. In order to evaluate the in vivo effect of adenosine, increasing concentrations (6, 60, 600 microg) of adenosine or prostaglandin E1 (PGE1) (10 microg) were injected into the corpora cavernosa of four healthy volunteers. Blood flow and erectile response were evaluated at different times by duplex sonography and visual inspection, respectively. It was found that adenosine increased cavernosal peak blood flow velocity in a time- and dose-dependent manner. The highest concentrations of injected adenosine elicited a response that was not statistically different from that of PGE1 (10 microg). However, in contrast to PGE1, a full or partial erection was never obtained. To further investigate the lack of effect of adenosine on penile tumescence (despite the substantial increase in cavernosal blood flow), in vitro experiments were performed on human deep dorsal penile veins (DDPV) obtained from surgical ligation for impotence. Adenosine did not affect basal tone, but it induced almost complete relaxation in noradrenaline-precontracted vein strips with an IC50=1.6 +/- 0.22 mM. Conversely, PGE1 stimulated a sustained increase in basal tone. Therefore, the lack of effect of adenosine on penile tumescence could be due to a simultaneous relaxing activity on penile corpora cavernosa and veins. In conclusion, our study indicates that adenosine relaxes HCC as well as penile veins without affecting erection, at least at the concentrations we have used. Conversely, PGE1 relaxes corpora cavernosa as well as adenosine but strongly stimulates vein contraction, allowing penile tumescence.

Adenosine↗
PubMed2000

Androgen receptor expression in prostate carcinoma cells suppresses alpha6beta4 integrin-mediated invasive phenotype.

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of alpha6beta4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of alpha6 and beta4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of alpha6beta4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further reduced alpha6 and beta4 messenger RNA expression as well as adhesion on laminin and Matrigel invasion. Our results indicate that androgens regulate cell-extracellular matrix adhesion and invasion by modulation of integrin expression and function, thus keeping a low invasive phenotype of prostate cancer cells.

Antigens, Surface↗
PubMed2000

Effect of a vitamin D3 analogue on keratinocyte growth factor-induced cell proliferation in benign prostate hyperplasia.

Prostate enlargement and function is under the dual control of androgens and intraprostatic growth factors. They regulate, in concert, prostate cell proliferation and apoptosis. An increased signaling of both growth factors and androgens are supposed to underlie benign prostate hyperplasia (BPH), one of the more common disorders of the aging male. Since, in clinical practice, androgen ablation resulted in a rather limited decrease in prostate volume, therapeutic strategies targeting intraprostatic growth factors are emerging. The activated form of vitamin D, vitamin D3, and some of its analogues have been described as potent regulators of cell growth and differentiation. In this study, we report the effects of one of these vitamin D3 analogues, 1,25-dihydroxy-16ene-23yne D3, or analogue (V), on the fate of isolated epithelial cells derived from patients with BPH. We essentially found that analogue (V), as well as vitamin D3, inhibited BPH cell proliferation and counteracted the mitogenic activity of a potent growth factor for BPH cells, such as keratinocyte growth factor (KGF). Moreover, analogue (V) induced bcl-2 protein expression, intracellular calcium mobilization, and apoptosis in both unstimulated and KGF-stimulated BPH cells. Since a short-term (5-min) incubation with analogue (V) reduced the KGF-induced tyrosine phosphorylation of a 120-kDA protein, corresponding to the KGF receptor, a rapid and direct cross-talk between these two molecules is suggested. Such a rapid effect of analogue (V), together with the transient induction of intracellular calcium waves, seems to indicate the partial involvement of a membrane, nongenomic receptor for vitamin D3. In conclusion, we demonstrated the antiproliferative and proapoptotic effect of analogue (V) in BPH cells and speculated on its possible use in the therapy of BPH.

Blotting, Western↗
PubMed2000

Erectile dysfunction: from biochemical pharmacology to advances in medical therapy.

Research on penile smooth muscle physiology has increased the number of drugs available for treating erectile dysfunction (ED). Penile erection involves the relaxation of smooth muscle in the corpus cavernosum. The key mediator of smooth muscle relaxation is nitric oxide (NO), which acts by increasing the cellular level of cGMP. Another cyclic nucleotide, cAMP, is involved in smooth muscle cell relaxation; cAMP formation is stimulated by a number of compounds, such as alprostadil. An increase in cAMP and/or cGMP levels can also be induced by inhibition of phosphodiesterases (PDEs), the enzymes involved in cyclic nucleotide breakdown. Both papaverine and sildenafil are PDE inhibitors. Papaverine is a non-specific inhibitor of these enzymes; sildenafil is an orally active, potent and selective inhibitor of GMP-specific PDE5, the predominant isoenzyme metabolizing cGMP in the cells of the corpus cavernosum. Penile smooth muscle contraction, induced by adrenergic fibers through alpha(1) adrenoceptors, produces detumescence, thus making alpha adrenoceptor antagonists suitable for maintenance of penile erection. The orally active drug yohimbine is a mixed alpha(1)-alpha(2) adrenoceptor antagonist that works by a dual mechanism; it facilitates sexual arousal by acting on alpha(2) adrenoceptors in the central nervous system and blocks adrenergic influences at peripheral level.

Adrenergic alpha-Antagonists↗
PubMed2000

[Doppler color ultrasonography in the diagnosis of erectile dysfunction of vascular origin].

The aim of this paper is to evaluate whether echo-color Doppler diagnostics allows to detect the different evolutionary stages of arteriopathy affecting the cavernous arteries of the penis. 124 patients with erectile deficiency were examined. All of them were evaluated by andrologic and psychologic anamnesis. An echo-color Doppler examination was performed on cavernous arteries before (basal) and after (dynamic) an injection of Prostaglandin E1 (10 micrograms). The penile circulation has been observed for twenty minutes after the injection to detect anomalous collateral vessels of the cavernous arteries. The population was divided into 4 groups according to the basal and dynamic peak systolic velocity (PSV). Cardiovascular risk factor incidence in patients with normal basal and dynamic PSV values (group A) and in patients with pathologic basal and dynamic PSV values (group C) is 10% and 44% respectively (p < 0.002). Psychologic disorder in group A and C is 41% and 26% respectively (p < 0.05). Patients with pathologic basal PSV and normal dynamic PSV (group B) show an intermediate (26%) incidence of cardiovascular disorders as compared to A and C. Anomalous side branches are equally represented in A and B (41% and 38%) and to a lesser extent in C (26%). Based on anamnestic and instrumental data, Group B ranges at an intermediate position between A and C. In this group, the presence of incipient arteriopathy affecting the penile circulation may be suspected. The ability to detect patients with vascular diseases still at a subclinical stage, as it already happens for other organs, would allow a prompt therapeutic action.

Adult↗
PubMed1999

State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii.

The relationship between state transitions and the kinetic properties of the electron transfer chain has been studied in Chlamydomonas reinhardtii. The same turnover rate of cytochrome f was found in state 1 and 2. However, while DBMIB was inhibitory in both states, DCMU was effective only in state 1. These observations suggest that linear electron transport was active only in state 1, while a cyclic pathway around photosystem (PS) I operated in state 2. The reversible shift from linear to cyclic electron transport was modulated by changes of PSII antenna size, which inactivated the linear pathway, and by oxygen, which inhibited the cyclic one. Attainment of state 2, under anaerobiosis in the dark, was associated with the decline of the ATP/ADP ratio in the cells and the dark reduction of the intersystem carriers. Upon illumination of the cells, the ATP/ADP ratio increased in a few seconds to the aerobic level. Then, several minutes later, the F(m) returned to the state 1 level, and O(2) evolution was reactivated. This suggests that ATP, though required for photosynthesis, is not the rate-limiting factor in the reactivation of photosynthetic O(2) evolution, which is rather controlled by the redox state of the electron carriers.

Adenosine Diphosphate↗
PubMed1999

Effects of inhibitors on the activity of the cytochrome b(6)f complex: evidence for the existence of two binding pockets in the lumenal site.

The effects of two inhibitors of electron transfer in the cytochrome b(6)f complex have been studied in whole cells of Chlamydomonas reinhardtii. DNP-INT affected equally the two steps of the concerted oxidation of plastoquinol at the Q(o) site; it decreased the rates of both cytochrome f reduction and cytochrome b(6) turnover, without affecting the amplitude of their redox signals. On the contrary, DBMIB inhibited only the rate of cytochrome f reduction while reducing, at the same time, the amplitude of cytochrome b(6) signals. The accessibility of DNP-INT to the Q(o) site was unaffected by preillumination, while that of DBMIB was greatly enhanced, even after a single turnover of the cytochrome b(6)f complex. Similar results were obtained with a mutant strain (FUD2) where the Q(o) site has an affinity for plastoquinol that is diminished by a factor of approximately 50 [Finazzi, G., et al. (1997) Biochemistry 36, 2867-2874]. However, the binding of the two inhibitors was differentially influenced by the mutation: a factor of approximately 250 was calculated for DNP-INT and a factor of only approximately 5 for DBMIB. This suggests that they bind within the Q(o) site in two distinct pockets, which are differentially involved in the process of quinol oxidation, in agreement with a recent model where two distinct positions for the reduced and semireduced quinones are considered [Crofts, A. R., and Berry, E. A. (1998) Curr. Opin. Struct. Biol. 8, 501-509].

Animals↗
PubMed1999

Screening for microdeletions of Y chromosome genes in patients undergoing intracytoplasmic sperm injection.

The potential of assisted reproduction techniques to transmit genetic defects causing male infertility raises questions concerning the need for a systematic genetic screen and counselling. Deletions of the long arm of the Y chromosome are frequently associated with a failure of spermatogenesis. The search for Y specific sequences and for the gene families RNA binding motif (RBM) and deleted in azoospermia (DAZ) have been introduced in many laboratories. The incidence of Y microdeletions varies widely between studies, from 1-55%. These differences are mainly related to study design. The highest incidence of microdeletions has been reported in well selected idiopathic azoospermic patients. Since microdeletions have been reported also in non-idiopathic patients, it is important to define what is the deletion frequency in unselected patients. We report Y chromosome microdeletion screening in 134 unselected patients undergoing intracytoplasmic sperm injection (ICSI). In the first part of the study we tested six Y chromosome markers. We found three patients with microdeletions (2.2%). Subdivision of the study population revealed a deletion incidence of 4.7% in azoospermic/cryptozoospermic patients; an incidence of 7% in idiopathic patients and an incidence of 16% in idiopathic azoospermic/cryptozoospermic patients. The second part of the study consisted of a screen for the presence of the Y chromosome genes, DBY, CDY, XKRY, eIF-1A, DAZ and BPY2. No additional gene-specific deletions were found. Further data on gene specific screening are needed especially for selected idiopathic patients.

Base Sequence↗
PubMed1999

Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects.

The presence of a novel functional estrogen receptor on the human sperm surface has been demonstrated by using different experimental approaches. Ligand blot analysis of sperm lysates, using peroxidase-conjugated estradiol as probe, identified a specific estradiol-binding protein of approximately 29-kDa apparent molecular mass. The same protein band was also revealed by using alphaH222 antibody, which is directed against the steroid binding domain of the genomic estrogen receptor. The biological effects of estrogen receptor were investigated by analyzing calcium fluxes, tyrosine phosphorylation, and acrosome reaction (AR) in response to 17beta-estradiol (17betaE2) and by measuring the steroid influence on calcium and AR in responses to progesterone (P), a well-known physiological stimulus for human spermatozoa. Our results demonstrate that 17betaE2 induces a rapid and sustained increase of intracellular calcium concentrations ([Ca2+]i). This effect is totally dependent on the presence of extracellular calcium, because it is completely abolished in a calcium-depleted medium. The dose-response curve for calcium increase to 17betaE2 is biphasic with a first component in the nanomolar range (effective concentration 50 = 0.60 +/- 0.12 nmol/L) and a second component in the micromolar range (EC50 = 3.80 +/- 0.26 micromol/L). 17BetaE2 stimulates tyrosine phosphorylation of several sperm proteins, including the 29-kDa protein band, and determines a reduction of calcium response to P, finally resulting in inhibition of P-stimulated sperm AR. Conversely, no direct effect of 17betaE2 is observed on AR. 17BetaE2 effects on calcium are clearly mediated by a membrane receptor, because they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E2 and reduced by sperm preincubation with alphaH222 antibody. Taken together, our results clearly show the presence of a functional surface estrogen receptor, of 29 kDa, on human spermatozoa. This receptor may play a role in the modulation of nongenomic action of P in these cells during the process of fertilization.

Acrosome Reaction↗
PubMed1999

Effects of progesterone on human spermatozoa: clinical implications.

Progesterone is a physiological stimulus of human sperm acrosome reaction. The effects of the steroid, which is present in high levels in the cumulus matrix that surrounds the oocyte, are mediated by an increase of intracellular calcium concentrations, tyrosine phosphorylation of proteins, efflux of chloride and stimulation of activity of phospholipases. These effects are due to activation of a nongenomic pathway. Two different types of receptors for progesterone, distinct from the genomic ones, have been identified on the surface of human spermatozoa. We demonstrated that sperm responsiveness to progesterone is impaired in subfertile patients and that is strictly correlated to the ability of fertilize the oocyte. In addition, the determination of sperm responsiveness is predictive of fertilizing ability with a positive predictive value of 90% and can be clinically useful for the preliminary assessment of the male partner to select the appropriate assisted reproductive technique.

Acrosome Reaction↗