Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.     

Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Dose-response for retinoic acid-induced forelimb malformations and cleft palate: a comparison of computerized image analysis and visual inspection

BACKGROUND: The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice. METHODS: Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis. RESULTS: Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg). CONCLUSIONS: Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289-295, 2004. Copyright 2004 Wiley-Liss, Inc.     

Histological changes induced in developing limb buds of C57BL mouse embryos submitted in utero to the combined influence of acetazolamide and cadmium sulphate

Microscopic defects in limb buds of C57BL mouse embryos after the combined teratogenic action of acetazolamide plus cadmium sulphate administered on day 9 of gestation were studied in serial sections. Postaxial deficiencies observed in 12-15-day embryos and affecting preferentially the right forelimbs were classified in nine morphological types according to increasing amounts of missing parts. Type X defect consists of a nearly complete amelia in which all four limbs are represented only by the girdle and proximal end of the stylopod. Type XI abnormality appears as an intermediate reduction affecting the area of digit IV. In addition to modifications of the forelimb bud shape detected from the 10-day stage onwards, observations made 24 and 48 hr after treatment confirmed that the postaxial defects result from an absolute lack of postaxial mesoderm occurring without cell necrosis as a consequence of a postaxial shortening of the apical ectodermal ridge (aer). In 10-day embryos, the latter appears shortened and hypertrophied; it is later fragmented into alternate thick and thin portions in 11-day affected limb buds. These ectodermal changes might account for the genesis of all types of defects observed. Untreated 9-day embryos with 12-25 pairs of somites display a number of asymmetries between their right and left forelimb territories: Until the 19-somite stage, the vascular supply to that area is provided exclusively by the umbilical vein, which is larger on the right side; the initial amount of somatopleural limb mesoderm is greater in the right rudiment and the genesis of its aer is slightly protracted as compared to the left one. These asymmetries might contribute to the right side predominance of the forelimb defects induced by acetazolamide and cadmium.     

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.     

New in vitro procedures for experimental studies on the development of 11-day mouse embryo forelimb buds

A new method has been developed for culturing 11-day mouse forelimb buds in vitro. In cultures performed with conventional procedures, skeletal pieces frequently appeared distorted and reduced in size. Moreover, forelimb buds explanted from embryos younger than a stage corresponding to 50 pairs of somites developed narrow hand plates devoid of radiated autopods. By contrast, in the new procedure using media supplemented with fetal calf serum and growth factors and enhancing distal feeding with carrier implants of catgut, enlarged pads were obtained that exhibited at least 4 digital rays in buds explanted from embryos with 40-44 pairs of somites. Compared with conventional procedures, the mean value of DNA content per limb bud was twice as great with use of our improved method. The ability of limb bud cells to proliferate and differentiate when cultured either in classical or in modified conditions, and the importance of the technical procedures, are discussed in the new prospect of in vitro developmental studies.     

Effects of Ethylene Glycol Monomethyl Ether and Its Metabolite, 2‐Methoxyacetic Acid,on Organogenesis Stage Mouse Limbs In Vitro

Exposure to ethylene glycol monomethyl ether (EGME), a glycol ether compound found in numerous industrial products, or to its active metabolite, 2‐methoxyacetic acid (2‐MAA), increases the incidence of developmental defects. Using an in vitro limb bud culture system, we tested the hypothesis that the effects of EGME on limb development are mediated by 2‐MAA‐induced alterations in acetylation programming. Murine gestation day 12 embryonic forelimbs were exposed to 3, 10, or 30 mM EGME or 2‐MAA in culture for 6 days to examine effects on limb morphology; limbs were cultured for 1 to 24 hr to monitor effects on the acetylation of histones (H3K9 and H4K12), a nonhistone protein, p53 (p53K379), and markers for cell cycle arrest (p21) and apoptosis (cleaved caspase‐3). EGME had little effect on limb morphology and no significant effects on the acetylation of histones or p53 or on biomarkers for cell cycle arrest or apoptosis. In contrast, 2‐MAA exposure resulted in a significant concentration‐dependent increase in limb abnormalities. 2‐MAA induced the hyperacetylation of histones H3K9Ac and H4K12Ac at all concentrations tested (3, 10, and 30 mM). Exposure to 10 or 30 mM 2‐MAA significantly increased acetylation of p53 at K379, p21 expression, and caspase‐3 cleavage. Thus, 2‐MAA, the proximate metabolite of EGME, disrupts limb development in vitro, modifies acetylation programming, and induces biomarkers of cell cycle arrest and apoptosis     

"Chemical surgery" as an approach to study morphogenetic events in embryonic mouse limb

Cytosine arabinoside (Ara-C) or retinoic acid (RA) was injected into pregnant mice in doses which induce a high incidence of limb defects. Within 4 hr of the treatment, extensive cell death was observed in the embryonic limb buds. However, the location of necrotic cells and the eventual limb defects were different for the two chemicals. Ara-C killed cells in those regions of the limb which were undergoing active proliferation. RA, on the other hand, had no effect on actively dividing cells but was lethal to cells of chondrogenic lineage at stages when their proliferation rate had fallen 7- to 10-fold below the original rate. In all cases, an excellent correlation between the location of dead cells (as seen 4 hr after drug treatment) and the eventual bony defects (as seen in the term fetuses) was observed. The unique properties of Ara-C and RA have been exploited in determining the relative levels of cytodifferentiation in the embryonic mouse limb buds. It is concluded that in the limbs of early 11th day mouse embryos (comparable to chick stage 19–20), differentiation of future skeletal elements has not yet begun. However, by the 12th day (comparable to chick stage 23), cell populations destined to form most of the future cartilages (except for digits) have already been established.     

Retinoid teratogenicity in the macaque: verification of dosing regimen

To further define teratogenicity associated with 13-cis-retinoic acid (13-cis-RA) in the cynomolgus monkey, the drug was orally administered on three different treatment regimens. Experiment (Exp.) 1 (2.5 mg/kg/day, gestational day [GD] 12-27, n = 11) investigated the teratogenicity of a single daily dose of 13-cis-RA administered shortly after embryo implantation. Pharmacokinetic sampling was done to determine retinoid profiles on the first (GD12) and last (GD27) days of treatment. Exposure to 13-cis-RA during early organogenesis in Exp. 2 (2.5 mg/kg/day, GD20-27, and 2 x 2.5 mg/kg/day, GD28-30, n = 5) investigated the potential adverse effects of 13-cis-RA on the developing limb. The use of multiple doses of 13-cis-RA in Exp. 3 (2 x 2.5 mg/kg/day, GD26-27, n = 5) investigated the necessity of double dosing on the induction of retinoid embryopathy in the macaque. Malformations of retinoid target organs as well as embryolethality were most prevalent when single daily doses of 13-cis-RA were administered during pre- and early organogenesis in Exp. 1. Moreover, multiple doses on GD26-27 failed to induce any manifestation of abnormal development in Exp. 3. These results confirm that the lowest observed adverse effect level (LOAEL) in macaques is 2.5 rather than 5.0 times greater than that observed in human pregnancies. Exposure during forelimb development (GD20-30) in Exp. 2 was unsuccessful in inducing defects of this skeletal region, although defects in several retinoid target organs (i.e., cerebellum and internal ear) were present, indicating that a teratogenic threshold was achieved. Pharmacokinetic analysis of 13-cis-RA and its metabolites on GD12 and 27 in Exp. 1 showed considerable exposure to the administered drug and its 4-oxo-metabolite. In contrast, the exposure to all-trans-RA was negligible. The results support the use of a specific treatment schedule in early gestation in the macaque as the most appropriate model for characterizing the teratogenic potential of retinoids in humans.     

HeLa细胞热休克蛋白的代谢动力学

 HeLa细胞经45℃,15min应激后,可诱导一组分子量为100,85,73,70,54kD的热休克蛋白,其中分子量为73/70kD的HSP产量最高。在产生HSP的同时,正常蛋白质合成受到抑制,并在随后数小时内恢复。HSP73/70在应激后能迅速诱导产生,应激后4—6小时为其合成高峰,10小时后明显减少,24小时恢复正常。其分解遵循指数规律,半衰期为49.9小时。     

Toxoplasma gondii-derived heat shock protein 70 induces lethal anaphylactic reaction through activation of cytosolic phospholipase A2 and platelet-activating factor via Toll-like receptor 4/myeloid differentiation factor 88

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce IFN-gamma-dependent lethal anaphylactic reaction in T. gondii-infected mice through an alternative PAF-mediated pathway, but not the classical immunoglobulin (Ig)E-dependent pathway. Although marked IFN-gamma production was observed by CD11b(+), CD11c(+), CD4(+) and CD8(+) splenocytes, CD11b(+) and CD11c(+) cells were shown to be the key effecter cells which generated pro-inflammatory lipid such as PAF and caused T.g.HSP70-induced anaphylactic reaction. In the present study, we found that the T.g.HSP70-induced anaphylactic reaction was not observed in TLR 4-deficient ((-/-)) mice, whereas it was observed in WT and TLR2(-/-) mice. The mRNA expression of PAF-AH, the main enzyme for PAF degradation, increased in T. gondii-infected WT and TLR2(-/-) but not in TLR4(-/-) mice after T.g.HSP70 injection. Furthermore, phosphorylation of cPLA(2), which is the key enzyme for pro-inflammatory lipid generation, was detected in CD11b(+) splenocytes of WT and TLR2(-/-) mice but not in TLR4(-/-) mice. Subsequently, cPLA(2) activation was suppressed by inhibiting the TLR4-directed p38 and p44/42 MAPK pathways. However, T.g.HSP70-induced anaphylactic reaction was observed in TRIF(-/-) mice, but not in MyD88(-/-) mice. These findings indicate the cPLA(2) activated-PAF production via TLR4/MyD88-dependent, but not TRIF-dependent, signaling pathway in T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice.     

Hydroxylamine moiety of developmental toxicants is associated with early cell death: a structure-activity analysis

BACKGROUND: Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. METHODS: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. RESULTS: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. CONCLUSIONS: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.     

Differentially expressed genes associated with 5-Aza2′-deoxycytidine-induced hindlimb defects in the Swiss Webster Mouse

5-Aza-2′-deoxycytidine (d-AZA) inhibits methylation of DNA, a process that serves as an epigenetic regulator of gene expression. We have shown that d-AZA causes temporally related defects in mice. Gestational day (GD) 10 treatment induced severe long-bone defects of the hindlimb but not the forelimb. Exposure of younger embryos (GD 8 or 9) does not induce similar defects in forelimbs. This limb-dependent response suggests that methylation alterations in genes specific for fore- or hindlimbs may contribute to the observed pattern of defects. Subtraction hybridization (SH) studies were conducted to identify differential expression of DNA subsequent to the administration of d-AZA to mice on GD 10. Hindlimb buds collected from both treated and untreated embryos at 4, 12, and 24 hours post-treatment were used. A clone isolated from the untreated sample (down-regulation in treated tissue) was identified as a member of the murine B1 family of repetitive sequences. The two other clones isolated from the treated tissue (up-regulation) were homologous to avian myogenic regulatory protein mRNA and activin receptor type II gene. Both species are active during embryogenesis. These findings suggest that the isolated clones may have roles in abnormal embryonic development when inappropriately expressed. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 135–141, 1998     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.     

In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart

BACKGROUND: Maternal diabetes exposes embryos to periods of hyperglycemia. Glucose is important for normal cardiogenesis, and Glut-1 is the predominant glucose transporter in the embryo. METHODS: Pregnant mice were exposed to 6 or 12 hr hyperglycemia during organogenesis using intraperitoneal (IP) injections of D-glucose on gestational day (GD) 9.5 (plug = GD 0.5). Embryos were examined for morphology and total cardiac protein, and embryonic hearts were evaluated for Glut-1 protein and mRNA expression immediately after treatment (GD 9.75, GD 10.0), as well as on GD 10.5 and GD 12.5. RESULTS: IP glucose injections were effective in producing sustained maternal hyperglycemia. Maternal hyperglycemia for 6 or 12 hr on GD 9.5, followed by normoglycemia, produced a decrease in overall size and total cardiac protein in embryos evaluated on GD 10.5 but no difference on GD 12.5. Cardiac Glut-1 expression was immediately upregulated in embryos exposed to 6 or 12 hr maternal hyperglycemia. On GD 10.5, cardiac Glut-1 expression was not different in embryos exposed to maternal hyperglycemia for 6 hr but was downregulated in embryos exposed for 12 hr. On GD 12.5, cardiac Glut-1 expression in embryos exposed to maternal hyperglycemia on GD 9.5 for 6 or 12 hr, followed by normoglycemia, was not different from controls. The temporal pattern was the same for Glut-1 protein and mRNA expression. CONCLUSIONS: Hyperglycemia-induced alterations in Glut-1 expression likely interfere with balance of glucose available to the embryonic heart that may affect cardiac morphogenesis.     

Placental improvement and reduced distal limb defects by maternal interferon-gamma injection in methylnitrosourea-exposed mice

BACKGROUND: Methylnitrosourea (MNU), an alkylating agent derived from creatinine metabolism, is cytotoxic, genotoxic, and mutagenic. Mid-gestational exposure to MNU leads to distal limb defects in mice. Previous studies have shown that nonspecific maternal immune stimulation protects against MNU-induced teratogenesis. A role for immune-mediated placental improvement in this effect remains uncertain. METHODS: The immune system of timed-pregnant C57BL/6N and CD-1 mice was stimulated by GD 7 intraperitoneal (IP) injection with the cytokine interferon-gamma (IFN-gamma). A teratogenic dose of MNU was then administered by IP injection on the morning of GD 9 to disrupt distal limb formation. Fetal limb length, body length, digital deformities, and placental integrity were evaluated on GD 14. RESULTS: The incidence of syndactyly, polydactyly, and interdigital webbing in MNU-exposed mice was decreased by maternal IFN-gamma treatment. In C57BL/6N mice, these defects were reduced by 47, 100, and 63%, respectively, as compared to previous reports on CD-1 mice, by 39, 71, and 20%, respectively. Administration of IFN-gamma significantly diminished MNU-induced endothelial and trophoblast placental damage in both strains of mice. CONCLUSIONS: These findings support a possible link between maternal immunity, placental integrity, and fetal distal limb development. Further, these results suggest that IFN-gamma might act through placental improvement to indirectly protect against MNU-induced fetal limb malformations.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo

BACKGROUND: Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). METHODS: In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. RESULTS: Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. CONCLUSIONS: In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.     

Alfalfa-derived HSP70 administered intranasally improves insulin sensitivity in mice

Heat shock protein (HSP) 70 is an abundant cytosolic chaperone protein that is deficient in insulin-sensitive tissues in diabetes and unhealthy aging, and is considered a longevity target. It is also protective in neurological disease models. Using HSP70 purified from alfalfa and administered as an intranasal solution, we tested in whether the administration of Hsp70 to diet-induced diabetic mice would improve insulin sensitivity. Both the 10 and 40 μg given three times per week for 26 days significantly improved the response to insulin. The HSP70 was found to pass into the olfactory bulbs within 4–6 hours of a single dose. These results suggest that a relatively inexpensive, plentiful source of HSP70 administered in a simple, non-invasive manner, has therapeutic potential in diabetes.