Effects and action mechanism of gonadotropins on ovarian follicular cells: A novel role of Sphingosine-1-Phosphate (S1P). A review.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control antral follicular growth by regulating several processes, such as the synthesis of hormones and signaling molecules, proliferation, survival, apoptosis, luteinization, and ovulation. To exert these effects, gonadotropins bind to their respective Gs protein-coupled receptors, activating the protein kinase A (PKA) pathway or recruiting Gq proteins to activate protein kinase C (PKC) signaling. Although the action mechanism of FSH and LH is clear, recently, it has been shown that both gonadotropins promote the synthesis of sphingosine-1-phosphate (S1P) in granulosa and theca cells through the activation of sphingosine kinase 1. Moreover, the inhibition of SPHKs reduces S1P synthesis, cell viability, and the proliferation of follicular cells in response to gonadotropins, and the addition of S1P to the culture medium increases the proliferation of granulosa and theca cells without apparent effects on sexual steroid synthesis. Therefore, we consider that S1P is a crucial signaling molecule that complements the canonical gonadotropin pathway to promote the proliferation and viability of granulosa and theca cells.

Short-term dietary concentrate supplementation during estrus synchronization treatment in beef cows increased IGF-I serum concentration but did not affect the reproductive response.

The objective of this study was to evaluate if short-term dietary concentrate supplementation increased IGF-I serum concentration and resulted in a reproductive response during estrus synchronization treatment in non-lactating beef cows. Thirty non-lactating beef cows (Bos indicus × Bos taurus) were allocated to the same pastureland and fed native tropical grasses as a basal diet. Cows were synchronized using a 7-day CO-Synch plus controlled internal drug release (CIDR) protocol and received fixed time artificial insemination (FTAI). Cows were divided into two groups; the control group (n = 16) received 0.5 kg of concentrate/cow/day, whereas the supplemented group (n = 14) received 4.0 kg of concentrate/cow/day. The period of supplementation was 10 days from the day of CIDR insert to FTAI. The concentration of IGF-I increased (P < 0.05) in the supplemented group, while no significant changes were observed in the control group. Moreover, at the time of insemination, IGF-I serum concentrations were higher in supplemented cows compared with control cows (P < 0.05). Notably, metabolite and insulin concentrations did not differ (P > 0.05) between treatment groups or sampling day. The response to estrus induction, measured as estrus presentation, ovulation rate, and pregnancy rate, was similar between experimental groups (P > 0.05). In conclusion, our results indicated that supplementation with dietary concentrate for 10 days in non-lactating beef cows changed the endocrine milieu, specifically increasing IGF-I serum concentration. However, these endocrine changes did not affect response to estrous induction treatment.

Sphingosine-1-phosphate, regulated by FSH and VEGF, stimulates granulosa cell proliferation.

Sphingosine-1-phosphate (S1P) is a bioactive polar sphingolipid which stimulates proliferation, growth and survival in various cell types. In the ovary S1P has been shown protect the granulosa cells and oocytes from insults such as oxidative stress and radiotherapy, and S1P concentrations are greater in healthy than atretic large follicles. Hence, we postulate that S1P is fundamental in follicle development and that it is activated in ovarian granulosa cells in response to FSH and VEGF. To test this hypothesis we set out: i) to evaluate the effect of FSH and VEGF on S1P synthesis in cultured bovine granulosa cells and ii) to analyse the effect of S1P on proliferation and survival of bovine granulosa cells in vitro. Seventy five thousand bovine granulosa cells from healthy medium-sized (4-7mm) follicles were cultured in 96-well plates in McCoy's 5a medium containing 10ng/mL of insulin and 1ng/mL of LR-IGF-I at 37°C in a 5% CO2/air atmosphere at 37°C. Granulosa cell production of S1P was tested in response to treatment with FSH (0, 0.1, 1 and 10ng/mL) and VEGF (0, 0.01, 0.1, 1, 10 and 100ng/mL) and measured by HPLC. Granulosa cells produced S1P at 48 and 96h, with the maximum production observed with 1ng/mL of FSH. Likewise, 0.01ng/mL of VEGF stimulated S1P production at 48, but not 96h of culture. Further, the granulosa cell expression of sphingosine kinase-1 (SK1), responsible for S1P synthesis, was demonstrated by Western blot after 48h of culture. FSH increased the expression of phosphorylated SK1 (P<0.05) and the addition of a SK1 inhibitor reduced the constitutive and FSH-stimulated S1P synthesis (P<0.05). Sphingosine-1-phosphate had a biphasic effect on granulosa cell number after culture. At low concentration S1P (0.1µM) increased granulosa cell number after 48h of culture (P<0.05) and the proportion of cells in the G2 and M phase of the cell cycle (P<0.05), whereas higher concentrations decreased cell number (10µM; P<0.05) by an increase (P<0.05) in the proportion of cells in apoptosis (hypodiploid cells). In addition, treatment with SK-178 suppressed the FSH- and VEGF-stimulated rise of the granulosa cells number (P<0.05). Interestingly, the effect of 0.1µM S1P on granulosa cell number and their proportion in G2/M phases is similar to that observed with 1ng/mL FSH. The results of this study are the first to demonstrate sphingosine-1-phosphate (S1P) synthesis in granulosa cells under the control of FSH and VEGF. The later achieved through the regulation of sphingosine kinase 1 expression. This S1P augments the proportion of cells in the G2/M phase of the cell cycle that translates in increased granulosa cell proliferation.

Reduction in the mRNA expression of sVEGFR1 and sVEGFR2 is associated with the selection of dominant follicle in cows.

The vascular endothelial growth factor (VEGF) is essential for follicular development by promoting follicular angiogenesis, as well as for the proliferation and survival of granulosa cells. The biological effects of VEGF are regulated by two membrane receptors, VEGFR1 and VEGFR2, and two soluble receptors, sVEGFR1 and sVEGFR2, which play an antagonistic role. Thus, the objective of this study was to identify the mRNA expression pattern of total VEGF, VEGFR1, VEGFR2, sVEGFR1 and sVEGFR2 in bovine preselected follicles (PRF) and post-selected follicles (POF). The mRNA expression of these five genes in both granulosa cells (GC) and theca cells (TC) was compared between follicles classified as PRF and POF based on their diameter and on their ratio of estradiol/progesterone (E2/P4). Results showed a lower expression of mRNA of sVEGFR1 and sVEGFR2 in POF than in PRF (p < .05). Regarding the mRNA expression of total VEGF, VEGFR1 and VEGFR2, there was no difference between POF and PRF follicles (p > .05). Our results showed that the mRNA expression of VEGFR2 and sVEGFR1 was more abundant than the expression of VEGFR1 and sVEGFR2, while GC was the main source of mRNA for total VEGF. On the other hand, TC was the follicular compartment where the receptors were most expressed. Our results suggest that non-dominant follicles maintain a greater concentration of the mRNA expression of both membrane and soluble VEGF receptors. On the other hand, follicular dominance is related to a reduction in the mRNA expression of sVEGFR1 and sVEGFR2, which may favour VEGF binding with VEGFR2 and, hence, improve the follicular health and development.

Climate change projected effects on coastal foundation communities of the Greater Everglades using a 2060 scenario: need for a new management paradigm.

Rising sea levels and temperature will be dominant drivers of coastal Everglades' foundation communities (i.e., mangrove forests, seagrass/macroalgae, and coral reefs) by 2060 based on a climate change scenario of +1.5 °C temperature, +1.5 foot (46 cm) in sea level, ±10 % in precipitation and 490 ppm CO2. Current mangrove forest soil elevation change in South Florida ranges from 0.9 to 2.5 mm year(-1) and would have to increase twofold to fourfold in order to accommodate a 2060 sea level rise rate. No evidence is available to indicate that coastal mangroves from South Florida and the wider Caribbean can keep pace with a rapid rate of sea level rise. Thus, particles and nutrients from destabilized coastlines could be mobilized and impact benthic habitats of southern Florida. Uncertainties in regional geomorphology and coastal current changes under higher sea levels make this prediction tentative without further research. The 2060 higher temperature scenario would compromise Florida's coral reefs that are already degraded. We suggest that a new paradigm is needed for resource management under climate change that manages coastlines for resilience to marine transgression and promotes active ecosystem management. In the case of the Everglades, greater freshwater flows could maximize mangrove peat accumulation, stabilize coastlines, and limit saltwater intrusion, while specific coral species may require propagation. Further, we suggest that regional climate drivers and oceanographic processes be incorporated into Everglades and South Florida management plans, as they are likely to impact coastal ecosystems, interior freshwater wetlands and urban coastlines over the next few decades.

First-in-man phase I trial of two schedules of the novel synthetic tetrahydroisoquinoline alkaloid PM00104 (Zalypsis) in patients with advanced solid tumours.

BACKGROUND: PM00104 binds guanines at DNA minor grooves, impacting DNA replication and transcription. A phase I study was undertaken to investigate safety, dose-limiting toxicities (DLTs), recommended phase II dose (RP2D), pharmacokinetics (PKs) and preliminary antitumour activity of PM00104 as a 1- or 3-h infusion three-weekly. METHODS: Patients with advanced solid tumours received PM00104 in a dose escalation trial, as guided by toxicity and PK data. RESULTS: A total of 47 patients were treated; 27 patients on the 1-h schedule (0.23-3.6 mg m(-2)) and 20 patients on the 3-h schedule (1.8-3.5 mg m(-2)). Dose-limiting toxicities comprised reversible nausea, vomiting, fatigue, elevated transaminases and thrombocytopenia, establishing the 1-h schedule RP2D at 3.0 mg m(-2). With the 3-h schedule, DLTs of reversible hypotension and neutropenia established the RP2D at 2.8 mg m(-2). Common PM00104-related adverse events at the RP2D comprised grade 1-2 nausea, fatigue and myelosuppression. In both schedules, PKs increased linearly, but doses over the 1-h schedule RP2D resulted in higher than proportional increases in exposure. A patient with advanced urothelial carcinoma had RECIST shrinkage by 49%, and three patients had RECIST stable disease ≥6 months. CONCLUSION: PM00104 is well tolerated, with preliminary evidence of antitumour activity observed. The 1-h 3-weekly schedule is being assessed in phase II clinical trials.

Molecular basis of dental sensitivity: The odontoblasts are multisensory cells and express multifunctional ion channels.

Odontoblasts are the dental pulp cells responsible for the formation of dentin. In addition, accumulating data strongly suggest that they can also function as sensory cells that mediate the early steps of mechanical, thermic, and chemical dental sensitivity. This assumption is based on the expression of different families of ion channels involved in various modalities of sensitivity and the release of putative neurotransmitters in response to odontoblast stimulation which are able to act on pulp sensory nerve fibers. This review updates the current knowledge on the expression of transient-potential receptor ion channels and acid-sensing ion channels in odontoblasts, nerve fibers innervating them and trigeminal sensory neurons, as well as in pulp cells. Moreover, the innervation of the odontoblasts and the interrelationship been odontoblasts and nerve fibers mediated by neurotransmitters was also revisited. These data might provide the basis for novel therapeutic approaches for the treatment of dentin sensibility and/or dental pain.

Alfalfa Root Flavonoid Production Is Nitrogen Regulated.

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.

Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles.

The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17ß-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In contrast, reduction of S1P and the S1P/CER in the antral follicle could trigger cellular death and atresia.

Surgical treatment of symptomatic colorectal endometriosis.

The approach to the treatment of bowel endometriosis has varied greatly. In this paper we present 77 consecutive patients with deep colorectal endometriosis treated with a full-thickness resection. Gynecologic procedures included conservative laparotomies for preserving fertility (39 patients); hysterectomy with bilateral salpingo-oophorectomy (29 patients); bilateral salpingo-oophorectomy (2 patients); left salpingo-oophorectomy (1 patient) and resection of pelvic endometriosis in patients with previous ablative surgery (6 patients). A low anterior bowel resection was performed in 68 patients (88.3%); a disc excision of the anterior rectal wall in 5 (6.5%); sigmoid resection in 3 (3.9%), and partial cecal resection in 1 (1.3%). The postoperative febrile morbidity was 10.4%, with no apparent anastomotic leaks. Of 33 patients who attempted to conceive postoperatively, 13 achieved a term pregnancy (39.4%). Complete relief of pelvic symptoms was obtained in 38 patients (49.4%); improvement in 30 (39%); no improvement in 8 (10.4%); and worsening of symptoms in 1 (1.2%). There has been no recurrence of symptomatic bowel endometriosis during 1 to 9 years of follow-up. Full-thickness resection of the colon for the treatment of deep bowel endometriosis is a safe procedure with low morbidity, good postoperative relief of symptoms, and favorable pregnancy rates.

Study and mathematical modeling of the production of propionic acid by Propionibacterium acidipropionici immobilized in a stirred tank fermentor.

A mathematical model was developed that describes production of propionic acid by fermentation of sweet whey with Propionibacterium acidipropionici immobilized in calcium polygalacturonate beads in a fermentor-type stirred tank. This mathematical model is constituted by a partial differential equations system, which fits consumption, production, growth and internal diffusion rates in the support. Fermentation was experimentally studied with free cells and immobilized cells, effective diffusivities of lactose and propionic acid were estimated in the support, and typical parameters of the model were obtained by nonlinear regression of the experimental data. The variance analysis shows that the combination of micro(max) and K(d) parameters is the source of variation most significative, also they were found to be the most sensitive parameters of the model. Finally, an effectiveness factor was calculated in order to assess the effect of mass transfer on the overall reaction rate observed.

Zero-temperature phase diagram of D2 physisorbed on graphane.

We determined the zero-temperature phase diagram of D2 physisorbed on graphane using the diffusion Monte Carlo method. The substrate used was C-graphane, an allotropic form of the compound that has been experimentally obtained through hydrogenation of graphene. We found that the ground state is the δ phase, a commensurate structure observed experimentally when D2 is adsorbed on graphite, and not the registered √3 x √3 structure characteristic of H2 on the same substrate.

A phase I pharmacokinetic study of PM00104 (Zalypsis) administered as a 24-h intravenous infusion every 3 weeks in patients with advanced solid tumors.

PURPOSE: PM00104 (Zalypsis) is a synthetic tetrahydroisoquinoline alkaloid with potent antiproliferative activity against tumor cell lines. This phase I study evaluated the safety, dose-limiting toxicities (DLTs), recommended dose for phase II trials (RD), pharmacokinetics (PK) and preliminary antitumor activity of PM00104 as a 24-h intravenous (i.v.) infusion every 3 weeks (q3wk). METHODS: Thirty-seven patients with refractory advanced solid tumors received PM00104 in a toxicity-guided dose escalation study design (3 + 3 patients per cohort). Plasma samples were collected for PK analysis. RESULTS: DLTs comprised severe neutropenia lasting >5 days (n = 4 patients), vomiting, thrombocytopenia, transaminase increases (n = 2 each), fatigue, tumor pain, myalgia, muscle stiffness, creatine phosphokinase increase and dosing delay >2 weeks due to moderate fatigue (n = 1 each). The RD was 4.0 mg/m(2). Most PM00104-related adverse events at the RD were mild or moderate; the most common were nausea, vomiting and fatigue. Myelosuppression and transaminase increases were transient and manageable. PK parameters increased linearly with dose. Higher PM00104 PK exposure was related to a decrease in hemoglobin, neutrophils, platelets and white blood cells. Area under the curve was directly correlated with both incidence and severity of nausea and vomiting. Three patients with hepatocellular carcinoma, esophageal adenocarcinoma and prostate adenocarcinoma had response evaluation criteria in solid tumors stable disease ≥3 months. CONCLUSIONS: PM00104 given as 24-h i.v. infusion q3wk has predictable and manageable toxicity, but resulted in more myelotoxicity (because of the higher dose level achieved as the RD) and a similar drug clearance compared to 1-h infusion schedules. Preliminary evidence of antitumor activity was observed.

Phase I study of weekly kahalalide F as prolonged infusion in patients with advanced solid tumors.

PURPOSE: Kahalalide F (KF) is a dehydroaminobutyric acid-containing peptide from marine origin with activity against several human malignant cell lines. This dose-escalating phase I clinical trial evaluated the maximum tolerated dose (MTD), and the recommended dose for further phase II studies (RD) of weekly KF given as a prolonged (3- to 24-h) intravenous (i.v.) infusion. METHODS: Eligible patients with advanced solid tumors and adequate performance status, hematologic, renal, and hepatic function were recruited into this study. RESULTS: A total of 106 patients were treated with KF at four different weekly schedules: 3-h (n = 40), 24-h (n = 59), and two transitional schedules [6-h (n = 4) and 12-h (n = 3)]. For the 3-h weekly schedule, the MTD was 1,200 µg/m² and the RD was 1,000 µg/m². For the 24-h weekly schedule, the MTD was reached (6,650 µg/m²), but the RD could not be confirmed. Asymptomatic and reversible grade 3/4 transaminase increase was the most common dose-limiting toxicity in both schedules. Fatigue, paresthesia, pruritus, nausea, vomiting, and rash were the most common KF-related adverse events. No major deviations from linearity were detected in the pharmacokinetic (PK) profiles of both schedules, which showed a narrow distribution and short body residence. Prolonged disease stabilization (≥3 months) occurred in eight patients: two with the 3-h schedule and six with the 24-h schedule. CONCLUSIONS: Administration of KF as prolonged weekly infusion appears feasible, with 3-h and 24-h infusion times having an acceptable safety profile.

A phase I and pharmacokinetic study of elisidepsin (PM02734) in patients with advanced solid tumors.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and recommended phase II dose (RD) of elisidepsin. METHODS: Eligible patients with refractory, advanced solid tumors received elisidepsin as 24-h intravenous infusion every 3 weeks. Pharmacokinetic profiles were analyzed during cycles 1 and 2. RESULTS: Forty-two patients received elisidepsin at doses from 0.5 to 6.8 mg/m(2). The MTD was 6.8 mg/m(2), and the RD was 5.5 mg/m(2). Cohort expansion at the RD was done at a fixed dose (FD) of 10 mg, considered equivalent to 5.5 mg/m(2). DLTs (reversible grade 3 transaminase increases) occurred at 6.8 mg/m(2) (n = 2 patients), 5.5 mg/m(2) (n = 1), and 10 mg FD (n = 1). One patient with esophageal adenocarcinoma achieved complete response for >38 months, and 12 patients had disease stabilization (8 for ≥3 months). Median time-to-progression for these 12 patients was 4.8 months. Plasma elisidepsin concentrations increased with dose. No drug accumulation between cycles was found. No correlation was observed between body surface area (BSA) and plasma clearance; therefore, elisidepsin was given as flat dose (in mg) in the expansion cohort at the RD and in ongoing clinical trials. CONCLUSIONS: Elisidepsin is well tolerated with predictable reversible transaminase increases. Encouraging preliminary evidence of antitumor activity was observed.

Molecular characterization and expression of alfalfa (Medicago sativa L.) flavanone-3-hydroxylase and dihydroflavonol-4-reductase encoding genes.

Flavonoids are plant phenolic compounds involved in leguminous plant-microbe interactions. Genes implied in the central branch (chalcone synthase (CHS), chalcone isomerase (CHI)) or in the isoflavonoid branch of the flavonoid biosynthesis pathway have been characterized in Medicago sativa. No information is available to date, however, on genes whose products are involved in the synthesis of other types of flavonoids. In this paper we present the genomic organization as well as the nucleotide sequence of one flavanone-3-hydroxylase (F3H) encoding gene of M. sativa, containing two introns and exhibiting 82-89% similarity at the amino acid level to other F3H proteins. This is the first report on the genomic organization of a f3h gene so far. We present also the sequence of a partial dihydroflavonol-4-reductase (DFR) M. sativa cDNA clone. Southern blot experiments indicated that f3h and dfr genes are each represented by a single gene within the tetraploid genome of M. sativa. By a combination of Northern blot and RT-PCR analysis, we showed that both f3h and dfr genes are expressed in flowers, nodules and roots, with a pattern distinct from chs expression. Finally, we show that dfr is expressed in M. sativa leaves whereas f3h is not. The role played by these two genes in organs other than flowers remains to be determined.

Rhizobium extracellular structures in the symbiosis.

The extracellular and surface polysaccharides produced by Rhizobium species constitute a composite macromolecular interface between the bacterial cell and its environment. Several of these polysaccharides are involved in the complex series of interactions leading to the establishment of an effective Rhizobium-legume symbiosis. Extracellular heteropolysaccharides (EPSs) are found in culture supernatants, while capsular polysaccharides adhere to the cell surface. Cyclic (1-2)-ß-d glucan is a periplasmic oligosaccharide that has also been found in the culture supernatants of some strains. The lipopolysaccharides (LPSs), which form part of the outer membrane and contain the O-somatic antigens, comprise the other major group of extracellular polysaccharides. In this review we will describe the major Rhizobium extracellular structures and their role in symbiosis with leguminous plants.