A blocking monoclonal antibody reveals dimerization of intracellular domains of ALK2 associated with genetic disorders.

Mutations in activin receptor-like kinase 2 (ALK2) can cause the pathological osteogenic signaling seen in some patients with fibrodysplasia ossificans progressiva and other conditions such as diffuse intrinsic pontine glioma. Here, we report that intracellular domain of wild-type ALK2 readily dimerizes in response to BMP7 binding to drive osteogenic signaling. This osteogenic signaling is pathologically triggered by heterotetramers of type II receptor kinases and ALK2 mutant forms, which form intracellular domain dimers in response to activin A binding. We develop a blocking monoclonal antibody, Rm0443, that can suppress ALK2 signaling. We solve the crystal structure of the ALK2 extracellular domain complex with a Fab fragment of Rm0443 and show that Rm0443 induces dimerization of ALK2 extracellular domains in a back-to-back orientation on the cell membrane by binding the residues H64 and F63 on opposite faces of the ligand-binding site. Rm0443 could prevent heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva that carries the human R206H pathogenic mutant.

Effect of whole blood clotting time in rats with ionized hypocalcemia induced by rapid intravenous citrate infusion.

Although the toxic effects of citrate including hemodynamic and cardiovascular changes result from a decrease in ionized calcium levels in serum due to chelating action, these effects of citrate on blood coagulation have not yet been fully clarified. The present study examines whether serum citrate and ionized calcium levels affect whole blood clotting time in rats using the test tube method in which citrate is administered by rapid intravenous infusion. Citrate was infused via the tail vein into 10 rats at 3, 4 or 5 mmol/kg/hr for 1 hr, and then whole blood clotting time, serum citrate and ionized calcium levels were determined. Whole blood clotting time did not significantly change at citrate infusion rates of 3 and 4 mmol/kg/hr. However, at 5 mmol/kg/hr, whole blood clotting time was significantly prolonged by a factor of 2.1 relative to the untreated group, when the serum citrate level was 10.03 +/- 1.39 mmol/l (59.0-fold higher than that in the untreated group) and the serum-ionized calcium level was 0.29 +/- 0.02 mmol/l (0.2-fold lower than that in the untreated group). These results suggest that whole blood clotting time is significantly prolonged in rats with severe ionized hypocalcemia.

Tolerable infusion rate of citrate based on clinical signs and the electrocardiogram in conscious dogs.

BACKGROUND & AIMS: The possible clinical significance of the toxic effects of citrate has not yet been fully clarified. This study was therefore conducted to confirm the toxicity and determine the tolerable infusion rate of citrate administered by rapid intravenous infusion to conscious dogs. METHODS: Citrate solutions were infused via the cephalic vein of 4 conscious dogs at 0.33, 0.67, or 1.33mmol/kg/h up to 1.33mmol/kg. Clinical signs and the electrocardiogram were observed during and after infusion. Serum citrate and ionized calcium levels were also measured. RESULTS: Although the mean citrate level increased in accordance with the infusion rate, the calcium level decreased. No significant changes in clinical signs or the electrocardiogram were observed during infusion at 0.33mmol/kg/h despite an increase in the serum citrate level to 1.22+/-0.11mmol/l (pre-infusion value: 0.38+/-0.01mmol/l) and a decrease in the serum calcium level to 1.28+/-0.03mmol/l (pre-infusion value: 1.50+/-0.05mmol/l). Vomiting and QTc prolongation were observed at 0.67mmol/kg/h or higher. Salivation and tachycardia were observed at 1.33mmol/kg/h. CONCLUSIONS: Based on clinical signs and the electrocardiogram, the tolerable infusion rate of citrate in conscious dogs is concluded to be 0.33mmol/kg/h.

Maximum acceptable infusion rate of citrate: relationship between blood ionized calcium levels and cardiovascular effects in anesthetized rats.

BACKGROUND & AIMS: Citrate is a useful chemical as a stabilizer for infusion solutions. However, cardiovascular depression associated with ionized hypocalcemia has been observed during massive transfusion of citrated blood products. The goal of the present study was to determine the maximum acceptable infusion rate of citrate and safe blood ionized calcium (Ca(2+)) levels. METHODS: Citrate was administered intravenously to anesthetized rats at infusion rates between 0.5 and 2.0 mmol/kg/h for 4 h. Changes in heart rate (HR), arterial blood pressure, and the concentrations of plasma citrate and blood Ca(2+) were measured. RESULTS: Infusion of citrate caused decreases in arterial blood pressure and HR, but no severe cardiovascular depression was observed at infusion rates up to 1.0 mmol/kg/h. Plasma citrate levels reached a steady state within 1 h after the start of infusion at up to 1.0 mmol/kg/h. The concentrations of plasma citrate and blood Ca(2+) were 1.35 and 0.89 mmol/l, respectively, 4h after the start of infusion at 1.0 mmol/kg/h. CONCLUSIONS: The maximum acceptable infusion rate of citrate was 1.0 mmol/kg/h in anesthetized rats, and no severe cardiovascular effects were observed when the blood Ca(2+) level was 0.89 mmol/l or above.

Infertility observed in female rats treated with N-acetyl-L-cysteine: Histopathological examination of ovarian follicles and recovery of fertility.

We previously reported infertility in female rats that received N-acetyl-L-cysteine (NAC) intravenously at a dosage of 1000 mg/kg/day. Unfertilized oocytes and gestation day 1 and 2 embryos were assessed morphologically, and the results suggested that absence or thinning of the zona pellucida (ZP) is related to infertility. However, the morphological characteristics of oocytes before ovulation and recovery from the effects of NAC were not clarified. In the present study, the ovarian follicles were histopathologically examined and the recovery of reproductive function was evaluated to investigate the effects of NAC. Female Sprague-Dawley rats at 10 weeks of age received NAC intravenously at 1000 mg/kg/day for more than 1 week. Thinning of the ZP was observed in the ovarian follicles in all stages of growth by light microscopy. Outflow of the components of the ZP between the corona radiata and disarrangement of the corona radiata were more pronounced in growing follicles than in large secondary follicles. Similar findings were observed by electron microscopy, and the effects of NAC were limited to the ZP. Infertility and thinning of the ZP were observed in the no-recovery NAC group, but not in the recovery NAC group, in which animals recovered within four estrous cycles after NAC administration. It has been reported that the ZP is expressed by oocytes or by both oocytes and granulosa cells, but no changes were noted in these cells. The present findings suggest that NAC affects the ZP directly and that reproductive function may recover from the effects of NAC.

Infertility observed in reproductive toxicity study of N-acetyl-L-cysteine in rats.

The toxic effects of i.v. administration of N-acetyl-l-cysteine (NAC), a component of parenteral nutrition solutions, on fertility and embryonic development were investigated in SD male and female rats at doses of 100, 300, and 1000 mg kg-1 day-1. Infertility was observed in females in the 1000-mg/kg group throughout the period from before mating to embryogenesis. No effect of NAC on the reproductive ability of the male rats was seen. The oocytes and embryos were assessed morphologically to clarify the cause of the effects of NAC. The unfertilized oocytes (UO) recovered from the ampullae of the uterine tubes and Gestational Day (GD) 1 and 2 embryos recovered from the oviducts or uterus of the rats that received NAC i.v. at a dosage of 1000 mg kg-1 day-1 for more than 1 wk before mating were assessed morphologically by stereomicroscopy. In addition, the thickness of the zona pellucida (ZP) was calculated by morphometric evaluation of the UO. Fewer UO were collected in the NAC group than in the control (nontreatment) group. Interestingly, ZP-lacking or partially ZP-lacking oocytes were observed in the NAC group, and the morphometric evaluation of the UO showed thinning of the ZP. The number of embryos in each animal was markedly decreased on GD1, and no embryos were recovered on GD2 in the NAC group. The oocytes that had ZP affected by NAC treatment were abnormal or nonviable. The findings of the present study suggest that changes in the ZP are related to the infertility associated with NAC.