Epidermal growth factor modulation of prostaglandins and nitrite biosynthesis in rat fetal membranes

The production of prostaglandins (PGs) and nitric oxide (NO) by amnion tissue may play a significant role in parturition. It is thought that epidermal growth factor (EGF) may be one of the fetal signals that governs the initiation of labor. The aim of the present study was to investigate the effect of EGF in vivo on the PGs and nitrite production of rat fetal membranes. We have evaluated the regulation of PGs and nitrite production in rat fetal membranes ex vivo. The intra-uterine administration of EGF 500 ng in day 21 of pregnancy induced increases in PGE(2) (P<0.001) and PGF(2alpha) (P<0.01) compared to the control fetal membranes from pregnant rats on day 22. Also, this dose of EGF diminished nitrate production significantly (P<0.01). We found that fetal membranes at term (days 18-22 of gestation) expressed EGF-R. The NO donor, nitroprussiate 300 and 600 microM, elicited an inhibitory effect on the PGE(2) and PGF(2alpha) stimulated synthesis. On the other hand, indomethacin 10(-6) and 10(-7)M, a non-selective cyclooxygenase inhibitor, reverted the inhibitory effect exerted by EGF. Hence, rat fetal membranes were found to express epidermal growth factor receptors and, under the effect of EGF, PGs and nitrites production pathways interact probably to prevent a toxic effect caused by an exacerbated synthesis of these mediators.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI2; PGD2; PGF2 and PGE2); BaCl2 or prostaglandin metabolites (15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha and 6-keto PGE1 in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl2, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha was displaced to the right of the curve for PGI2 and that for 6-keto-PGE1 to the left. It was also demonstrated that the uterine motility elicited by 10(-5) M PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha or BaCl2 fluctuated during the same period around more constant levels. The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI2, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Effects of general,specific and combined warm-up on explosive muscular performance

The purpose of this study was to compare the acute effects of general, specific and combined warm-up (WU) on explosive performance. Healthy male (n = 10) subjects participated in six WU protocols in a crossover randomized study design. Protocols were: passive rest (PR; 15 min of passive rest), running (Run; 5 min of running at 70% of maximum heart rate), stretching (STR; 5 min of static stretching exercise), jumping [Jump; 5 min of jumping exercises – 3x8 countermovement jumps (CMJ) and 3x8 drop jumps from 60 cm (DJ60)], and combined (COM; protocols Run+STR+Jump combined). Immediately before and after each WU, subjects were assessed for explosive concentric-only (i.e. squat jump – SJ), slow stretch-shortening cycle (i.e. CMJ), fast stretch-shortening cycle (i.e. DJ60) and contact time (CT) muscle performance. PR significantly reduced SJ performance (p =0.007). Run increased SJ (p =0.0001) and CMJ (p =0.002). STR increased CMJ (p =0.048). Specific WU (i.e. Jump) increased SJ (p =0.001), CMJ (p =0.028) and DJ60 (p =0.006) performance. COM increased CMJ performance (p =0.006). Jump was superior in SJ performance vs. PR (p =0.001). Jump reduced (p =0.03) CT in DJ60. In conclusion, general, specific and combined WU increase slow stretch-shortening cycle (SSC) muscle performance, but only specific WU increases fast SSC muscle performance. Therefore, to increase fast SSC performance, specific fast SSC muscle actions must be included during the WU.     

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.