Jeanne Shepshelovich

TNF‐α Protects Embryos Exposed to Developmental Toxicants

BACKGROUND:  Tumor necrosis factor α (ΤNF‐α) has been implicated in mediating post‐implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF‐α further, a comparative study was performed in TNF‐α knockout and TNF‐α positive mice, exposed to a reference teratogen, cyclophosphamide (CP).

Apoptosis in the Uterus of Mice with Pregnancy Loss

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis‐regulating gene products p53 and bcl‐2.

Immunostimulation increases the resistance of mouse embryos to the teratogenic effect of diabetes mellitus

Summary The present work was aimed to assess the possible effect of stimulation of the maternal immune system on the teratogenic potential of diabetes mellitus. ICR female mice were immunized with splenocytes of male rats 3 weeks before the beginning of mating and were injected with 240 mg/kg streptozocin (STZ) 10 days after immunization. Females with blood glucose levels over 27.8 mmol/l and HbA1 c levels over 6 standard deviations (SD) above the mean of intact animals were used for teratological studies. The rate of malformed fetuses, resorptions and fetal weights were evaluated for animals killed on day 19 of pregnancy using routine teratological methods. Also, phenotyping of spleen cells of these females was performed by fluorescein activated cell sorter analysis. Two main effects possibly due to immunostimulation of ICR females were observed: 1) immunostimulated females had significantly fewer litters with malformed fetuses than non-immunized females: only 4 litters out of 22 (18 %) compared to 10 out of 16 (63 %). Correspondingly, the incidence of malformed fetuses was also decreased: 2.1 compared to 8.9 %; 2) a significant increase in the pregnancy rate in immunized diabetic ICR mice: 69 % as compared to 44 % in non-immunized diabetic females. Also, immunostimulation resulted in a visible increase in spleen cellularity and a certain increase in the number of cells with mature T-cell and macrophage surface markers. These results strongly suggest that immunostimulation increases the tolerance of ICR females to the teratogenic effect of STZ-induced diabetes. [Diabetologia (1997) 40: 635–640]

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft-mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures-spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL-laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein-protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

NF-κB DNA-binding activity in embryos responding to a teratogen, cyclophosphamide

BackgroundThe Rel/NF-κB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-κB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFα-induced physiological apoptosis. This study assesses whether NF-κB may be involved in the embryos response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstraiting differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies).ResultsThe embryonic brain and liver were used as target organs. We observed that the Cyclophosphamide-induced excessive apoptosis in the brain, followed by the formation of severe craniofacial structural anomalies, was accompanied by suppression of NF-κB DNA-binding activity as well as by a significant and lasting increase in the activity of caspases 3 and 8. However, in the liver, in which cyclophosphamide induced transient apoptosis was not followed by dysmorphogenesis, no suppression of NF-κB DNA-binding activity was registered and the level of active caspases 3 and 8 was significantly lower than in the brain. It has also been observed that both the brain and liver became much more sensitive to the CP-induced teratogenic insult if the embryos were exposed to a combined treatment with the teratogen and sodium salicylate that suppressed NF-κB DNA-binding activity in these organs.ConclusionThe results of this study demonstrate that suppression of NF-κB DNA-binding activity in embryos responding to the teratogenic insult may be associated with their decreased resistance to this insult. They also suggest that teratogens may suppress NF-κB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion.

Genomic analysis of a heterogeneous Mendelian phenotype: multiple novel alleles for inherited hearing loss in the Palestinian population.

Recessively inherited phenotypes are frequent in the Palestinian population, as the result of a historical tradition of marriages within extended kindreds, particularly in isolated villages. In order to characterise the genetics of inherited hearing loss in this population, we worked with West Bank schools for the deaf to identify children with prelingual, bilateral, severe to profound hearing loss not attributable to infection, trauma or other known environmental exposure. Of 156 families enrolled, hearing loss in 17 families (11 per cent) was due to mutations in GJB2 (connexin 26), a smaller fraction of GJB2-associated deafness than in other populations. In order to estimate how many different genes might be responsible for hearing loss in this population, we evaluated ten families for linkage to all 36 known human autosomal deafness-related genes, fully sequencing hearing-related genes at any linked sites in informative relatives. Four families harboured four novel alleles of TMPRSS3 (988ΔA = 352stop), otoancorin (1067A > T = D356V) and pendrin (716T > A = V239D and 1001G > T = 346stop). In each family, all affected individuals were homozygous for the critical mutation. Each allele was specific to one or a few families in the cohort; none were widespread. Since epidemiological tests of association of mutations with deafness were not feasible for such rare alleles, we used functional and bioinformatics approaches to evaluate their consequences. In six other families, hearing loss was not linked to any known gene, suggesting that these families harbour novel genes responsible for this phenotype. We conclude that inherited hearing loss is highly heterogeneous in this population, with most extended families acting as genetic isolates in this context. We also conclude that the same genes are responsible for hearing loss in this population as elsewhere, so that gene discovery in these families informs the genetics of hearing loss worldwide.

Protein synthesis inhibitors and the chemical chaperone TMAO reverse endoplasmic reticulum perturbation induced by overexpression of the iodide transporter pendrin

An outcome of overloading of the endoplasmic reticulum (ER) folding machinery is a perturbation in ER function and the formation of intracellular aggregates. The latter is a key pathogenic factor in numerous diseases known as ER storage diseases. Here, we report that heterologous overexpression of the green fluorescent protein-tagged iodide transporter pendrin (GFP-PDS) perturbs folding and degradation processes in the ER. Pendrin (PDS) is a chloride-iodide transporter found in thyroid cells. Mutations in PDS can cause its retention in the ER and are associated with Pendred syndrome. Biochemical and live-cell analyses demonstrated that wild-type GFP-PDS is predominantly retained in perinuclear aggregates and in ER membranes, causing their collapse and vesiculation. Inhibition of protein synthesis by cycloheximide (CHX) or puromycin caused dissociation of the GFP-PDS aggregates and returned the ER to its normal reticular morphology. Blocking protein synthesis promoted folding and export of ER-retained GFP-PDS, as demonstrated by surface-biotinylation analysis and by CHX- or puromycin-induced accumulation of YFP-PDS in the Golgi apparatus during a 20°C temperature-block experiment. The chemical chaperone trimethylamine-N-oxide (TMAO) also reversed the GFP-PDS-mediated ER collapse and vesiculation, suggesting that exposed hydrophobic stretches of misfolded or aggregated GFP-PDS may contribute to ER retention. These data suggest that GFP-PDS is a slow-folding protein with a propensity to form aggregates when overexpressed. Thus, we describe a system for the reversible induction of ER stress that is based entirely on the heterologous overexpression of GFP-PDS.

Expression of Tumor Necrosis Factor-α in the Pregnant Uterus of Diabetic Mice: Effect of Maternal Immunopotentiation

PROBLEM: Tumor necrosis factor (TNF)‐α mRNA and protein are expressed in the pregnant uterus of streptozotocin‐induced diabetic mice at various stages of pregnancy. We intend to characterize their pattern and to evaluate whether the potentiation of the maternal immune system alters the pattern of the expression of the cytokine. 
METHOD OF STUDY: Diabetes was induced in ICR mice by streptozotocin injection. To modulate maternal immune responses, ICR mice were injected intrauterine with rat splenocytes 3 weeks before mating. The expression of TNF‐α mRNA and protein was evaluated by in situ hybridization and immunohistochemistry techniques. 
RESULTS. The population of diabetic mice used in this study demonstrated a reduction in pregnancy rate and an increased number of litters with severely malformed fetuses. It has been observed that these disturbances are associated with a clear increase in TNF‐α mRNA and protein expression in the uterus of these mice. Maternal immunopotentiation, while improving reproductive performance of these diabetic mice, was found to be accompanied by a reduced expression of TNF‐α, both at the mRNA and protein level. 
CONCLUSIONS. The results of the present study suggest a possible involvement of TNF‐α in mechanisms underlying diabetes‐associated dismorphogenesis. Normalization of TNF‐α expression by maternal immunopotentiation might represent a mechanism mediating its protective effect against diabetes‐induced embryotoxic insult.

Teratogen-induced apoptosis may be affected by immunopotentiation

Intra-uterine immunization of mice with paternal allogeneic or xenogeneic (rat) splenocytes was found to increase embryo tolerance to cyclophosphamide (CP)-induced teratogenesis. As the CP-induced teratogenic effect was shown to be associated with apoptosis, the present study was designed to investigate whether the protective effect of immunopotentiation may be realized via an alteration of CP-induced apoptosis. Various doses of CP were injected intraperitoneally into ICR mice on day 12 of pregnancy. Intra-uterine immunization with xenogeneic rat splenocytes was carried out 3 weeks before mating. Implantation sites, resorptions, live and dead fetuses, as well as soft tissue anomalies and external malformations, were recorded to evaluate the CP-induced embryotoxic effect. In parallel, flow cytometric analysis and DNA fragmentation assay were used for evaluation of CP-induced apoptosis in limbs, tail and whole embryos. The treatment of mothers with a high dose of CP induced the death of almost all embryos and striking fetal growth retardation in survivors. This strong embryotoxic effect was accompanied by very prominent DNA degradation in cells collected from whole embryos. Immunostimulation caused a dramatic decrease of embryonal loss (by approximately 50%) and a significant (about 30%) increase in fetal weight. Such an increase in fetal survival and in fetal weight was found to be accompanied by a clear decrease in apoptosis level in embryo cell population as judged by DNA gel electrophoresis with subsequent quantitation of DNA fragmentation in negatives by an image analysis technique. After treatment with a low dose of CP, a decrease in the proportion of fetuses with limb and tail anomalies in immunized females was accompanied by a decrease in the proportion of apoptotic nuclei in cells taken from limbs and tails. The results of this study suggest that the teratogen-induced apoptosis may, at least partly, be dependent on fetomaternal immune interactions.

Potentiation of the Maternal Immune System may Modify the Apoptotic Process in Embryos Exposed to Developmental Toxicants

PROBLEM: We have previously shown that teratogen‐induced embryonic maldevelopment may result from excessive apoptosis in affected organs, but the mechanisms underlying this process are not well understood. Here we investigate the ability of maternal immunopotentiation to affect the apoptotic process and its regulatory genes p53 and bcl‐2 in embryos exposed to a teratogenic insult.