Establishing life is a calcium-dependent TRiP: Transient receptor potential channels in reproduction

Calcium plays a key role in many different steps of the reproduction process, from germ cell maturation to placental development. However, the exact function and regulation of calcium throughout subsequent reproductive events remains rather enigmatic. Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. On the one hand, endometrial cell will undergo massive changes to support an implanting embryo, including stromal cell decidualization. On the other hand, trophoblast cells from the trophectoderm surrounding the inner cell mass will differentiate and acquire new functions such as hormone secretion, invasion and migration. The need for calcium in the different gestational processes implicates the presence of specialized ion channels to regulate calcium homeostasis. The superfamily of transient receptor potential (TRP) channels is a class of calcium permeable ion channels that is involved in the transformation of extracellular stimuli into the influx of calcium, inducing and coordinating underlying signaling pathways. Although the necessity of calcium throughout reproduction cannot be negated, the expression and functionality of TRP channels throughout gestation remains elusive. This review provides an overview of the current evidence regarding the expression and function of TRP channels in reproduction.     

Solid-phase synthesis of C-terminally modified peptides.

In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.     

Noonan-like phenotype in monozygotic twins with a duplication-deficiency of the long arm of chromosome 18 resulting from a maternal paracentric inversion

We report on newborn monozygotic twins with a Noonan-like phenotype, and multiple congenital anomalies due to a monocentric recombinant chromosome 18. The mother carried a paracentric inversion of the long arm of chromosome 18, inv(18)(q21.1q22.3). Cytogenetic, fluorescent in situ hybridization, comparative genomic hybridization and DNA marker analyses allowed the delineation of the deleted (18q22.3–qter) and duplicated (18q12.1–q21.1) chromosomal regions in the recombinant chromosome 18, and suggest that this duplication-deletion chromosome 18 resulted from breakage of a dicentric recombinant chromosome 18 with subsequent reconstitution of telomeric sequences on the long arm. Marked variability is observed in the phenotypic expression of the same chromosomal anomaly in these monozygotic twins. The clinical findings of these patients are compared with those reported in proximal 18q-duplication and distal 18q-deletion patients. The clinical features of both infants are compatible with Noonan syndrome, suggesting that a locus for this syndrome may be located on the long arm of chromosome 18. Received: 16 April 1998 / Accepted: 17 June 1998     

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.     

The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

Use of uterine artery Doppler ultrasonography to predict pre-eclampsia and intrauterine growth restriction: a systematic review and bivariable meta-analysis

Background

Alterations in waveforms in the uterine artery are associated with the development of pre-eclampsia and intrauterine growth restriction. We investigated the predictive accuracy of all uterine artery Doppler indices for both conditions in the first and second trimesters.

Methods

We identified relevant studies through searches of MEDLINE, EMBASE, the Cochrane Library and Medion databases (all records to April 2006) and by checking bibliographies of identified studies and consulting with experts. Four of us independently selected studies, extracted data and assessed study validity. We performed a bivariable meta-analysis of sensitivity and specificity and calculated likelihood ratios.

Results

We identified 74 studies of pre-eclampsia (total 79 547 patients) and 61 studies of intrauterine growth restriction (total 41 131 patients). Uterine artery Doppler ultrasonography provided a more accurate prediction when performed in the second trimester than in the first-trimester. Most Doppler indices had poor predictive characteristics, but this varied with patient risk and outcome severity. An increased pulsatility index with notching was the best predictor of pre-eclampsia (positive likelihood ratio 21.0 among high-risk patients and 7.5 among low-risk patients). It was also the best predictor of overall (positive likelihood ratio 9.1) and severe (positive likelihood ratio 14.6) intrauterine growth restriction among low-risk patients.

Interpretation

Abnormal uterine artery waveforms are a better predictor of pre-eclampsia than of intrauterine growth restriction. A pulsatility index, alone or combined with notching, is the most predictive Doppler index. These indices should be used in clinical practice. Future research should also concentrate on combining uterine artery Doppler ultrasonography with other tests.Pre-eclampsia and intrauterine growth restriction remain important causes of maternal and perinatal morbidity and mortality.^1–3 Maternal complications of pre-eclampsia include coagulopathy, renal and liver failure, and stroke.¹ Adults who were affected by intrauterine growth restriction in utero are at increased risk for cardiovascular disease, hypertension and type 2 diabetes.^4,5Pre-eclampsia and intrauterine growth restriction are characterized by abnormal placenta formation,⁶ which results in inadequate uteroplacental blood flow. This has led to the idea of using Doppler ultrasonography to assess the velocity of uterine artery blood flow as part of routine ultrasound screening.⁷ Low end-diastolic velocities and an early diastolic notch characterize the waveforms of uterine artery blood flow in women who are not pregnant or are in their first trimester. Persistence of a diastolic notch (beyond 24 weeks'' gestation) or abnormal flow velocity ratios have been associated with inadequate trophoblast invasion.⁸Accurate prediction of pre-eclampsia and intrauterine growth restriction is crucial to allow judicious allocation of resources for monitoring and preventive treatment to improve maternal and perinatal outcomes.^9,10 However, studies investigating the predictive accuracy of uterine artery Doppler indices (Box 1) have revealed considerably varied results. Thus, it is questionable whether uterine artery Doppler ultrasonography should be used as a predictive test. We undertook this review to investigate the accuracy of all uterine artery Doppler indices in predicting pre-eclampsia and intrauterine growth restriction.Open in a separate windowBox 1     

Preventive action of organosilicon treatments against disfigurement of wood under laboratory and outdoor conditions

Organosilicons and biocides with known effectiveness against fungal disfigurement were used for dipping or impregnating Scots pine sapwood specimens. All specimens were artificially or naturally weathered and the colour of all specimens was determined with a spectrophotometer at fixed times. After artificial weathering the specimens were used in blue stain tests according to EN 152 or according to the EN 152 reverse method. The naturally exposed specimens were inspected for fungal disfigurement on their back side. Although the results learn that the coating approach is far better than the wood preservatives approach for evaluating blue stain attack of organosilicon-treated wood, organosilicons fail to protect wood under laboratory conditions. Outdoor exposure, however, revealed that organosilicon impregnated specimens were better protected against fungal disfigurement. The addition of a biocide improves the performance. Artificially aged specimens did not show significant colour differences compared to untreated Scots pine sapwood, while naturally aged specimens did, depending on the treatment conditions and presence of biocides. Organosilicons are able to reduce leaching of (degraded) wood constituents, leading to fewer colour changes compared to untreated scots pine and to decreased availability of nutrients for superficial fungal growth.