Interaction of PDGFRA promoter haplotypes and maternal environmental exposures in the risk of spina bifida

BACKGROUND: Neural tube defects are multifactorial malformations involving both environmental exposures, such as maternal nutrition, and genetic factors. Aberrant expression of the platelet‐derived growth factor alpha‐receptor (PDGFRA) gene has been implicated in neural‐tube‐defect etiology in both mice and humans. METHODS: We investigated possible interactions between the PDGFRA promoter haplotype of mother and child, as well as maternal glucose, myo‐inositol, and zinc levels, in relation to spina bifida offspring. Distributions were determined of the PDGFRA promoter haplotypes H1 and H2 in a Dutch cohort, consisting of 88 spina bifida children with 56 of their mothers, and 74 control children with 72 of their mothers, as well as maternal plasma glucose, myo‐inositol, and red blood cell zinc concentrations. RESULTS: A significantly higher frequency of H1 was observed in children with spina bifida than in controls (30.1 vs. 20.3%; OR = 1.69, 95% CI 1.02–2.83). High maternal body mass index (BMI) and glucose were significant risk factors for both H1 and H2 children, whereas low myo‐inositol and zinc were risk factors for H2 but not for H1 children. Stepwise multiple logistic regression analysis showed that high maternal glucose and low myo‐inositol are the main risk factors for H2 spina bifida children, whereas for H1 spina bifida children, maternal BMI was the main risk factor. Interestingly, H1 mothers (median 165.5 cm) showed a significantly lower body height than H2 mothers (median 169.1 cm; p = 0.003). CONCLUSIONS: These data suggest that the child's PDGFRA promoter haplotype is differentially sensitive for periconceptional exposure to glucose, myo‐inositol, and zinc in the risk of spina bifida. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Quantification of ErbB3 receptor density on human breast cancer cells, using a stable radio-labeled mutant of NRG1beta

ErbB3 transactivation can make tumor cells resistance to ErbB1/ErbB2 targeting drugs. This urges for a reliable method to determine cell surface ErbB3 levels, but in our hands iodinated NRG1β is unstable and tends to underestimate the number of ErbB3 receptors in a radio-receptor assay. Here we show by the use of a radio-labeled high affinity neuregulin mutant NRG/YYDLL that ErbB3 levels can be determined in a reliable manner by Scatchard analysis. Furthermore we show by differential competition with unlabeled NRG/YYDLL and betacellulin that the number of ErbB3 and ErbB4 receptors can be quantified separately on cultured human breast cancer cells.     

Replacement of primary chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of avian leucosis viruses.

International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-detection, as alternative for primary CEF, was studied in three types of experiments: (1) in titration experiments without cell passage, (2) in experiments with passages in cell cultures according to European Pharmacopoeia requirements, and (3) in experiments with commercial live avian vaccines that had been spiked with known amounts of ALV. In all tests the sensitivity of ALV-A and ALV-J detections on DF-1 cells was at least as high as on primary CEF. The sensitivity of ALV-B detection was always superior when DF-1 cells were used. ALV were detected earlier in all comparative tests when DF-1 cells were used. ALV-A, ALV-B and ALV-J all induced CPE on DF-1 cells, whereas no clear CPE was seen on CEF-cells. For reasons of sensitivity, standardisation as well as reduction of animal use, the data support the use of DF-1 cells to monitor absence of ALV in vaccine virus seed lots or finished products.     

A new method for high yield purification of type beta transforming growth factor from human platelets

A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by lyophilization facilitate the quantification of the growth factor in biological assays. Based on immunological characterization the purified acid-stable, highly basic transforming growth factor beta is the beta 1 form. Using the present method pure platelet TGF beta 1 is obtained in very high yield. 40 units of outdated human platelets yield 800 micrograms pure TGF beta 1, which is about a 10-20 fold higher yield than reported for other purification procedures.     

Multi-generational evaluation of genetic diversity and parentage in captive southern pygmy perch (<Emphasis Type="Italic">Nannoperca australis</Emphasis>)

Maintaining genetic diversity within captive breeding populations is a key challenge for conservation managers. We applied a multi-generational genetic approach to the captive breeding program of an endangered Australian freshwater fish, the southern pygmy perch (Nannoperca australis). During previous work, fish from the lower Murray-Darling Basin were rescued before drought exacerbated by irrigation resulted in local extinction. This endemic lineage of the species was captive-bred in genetically designed groups, and equal numbers of F1 individuals were reintroduced to the wild with the return of favourable habitat. Here, we implemented a contingency plan by continuing the genetic-based captive breeding in the event that a self-sustaining wild population was not established. F1 individuals were available as putative breeders from the subset of groups that produced an excess of fish in the original restoration program. We used microsatellite-based parentage analyses of these F1 fish to form breeding groups that minimized inbreeding. We assessed their subsequent parental contribution to F2 individuals and the maintenance of genetic diversity. We found skewed parental contribution to F2 individuals, yet minimal loss of genetic diversity from their parents. However, the diversity was substantially less than that of the original rescued population. We attribute this to the unavoidable use of F1 individuals from a limited number of the original breeding groups. Alternative genetic sources for supplementation or reintroduction should be assessed to determine their suitability. The genetic fate of the captive-bred population highlights the strong need to integrate DNA-based tools for monitoring and adaptive management of captive breeding programs.     

Local induction of pacemaking activity in a monolayer of electrically coupled quiescent NRK fibroblasts

Cultures of normal rat kidney (NRK) fibroblasts may display spontaneous calcium action potentials which propagate throughout the cellular monolayer. Pacemaking activity of NRK cells was studied by patch clamp electrophysiology and vital calcium imaging, using a new experimental approach in which a ring was placed on the monolayer in order to physically separate pacemakers within or under the ring and follower cells outside the ring. Stimulation of cells inside the ring with IP(3)-generating hormones such as prostaglandin F(2alpha) (PGF(2alpha)) resulted in the induction of periodic action potentials outside the ring, which were abolished when the L-type calcium channel blocker nifedipine was added outside the ring, but not inside the ring. PGF(2alpha)-treated cells displayed asynchronous IP(3)-mediated calcium oscillations of variable frequency, while follower cells outside the ring showed synchronous calcium transients which coincided with the propagating action potential. Mathematical modelling indicated that addition of PGF(2alpha) inside the ring induced both a membrane potential gradient and an intracellular IP(3) gradient, both of which are essential for the induction of pacemaking activity under the ring. These data show that intercellular coupling between PGF(2alpha)-treated and non-treated cells is essential for the generation of a functional pacemaker area whereby synchronization of calcium oscillations occurs by activation of L-type calcium channels.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.