Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

Prediction of stage of pregnancy in prolific sheep using ultrasound measurement of fetal bones.

The use of ultrasound to estimate stage of pregnancy was assessed in 32 ewes of a prolific genotype carrying 7 singleton fetuses and 9 twin, 10 triplet and 6 quadruplet litters that were scanned on six occasions from 60 to 120 days of gestation. At least one ultrasound measurement per ewe of fetal metacarpal bone length (MCL), biparietal diameter (BPD), or of both bones was made on over 90% of attempts (n = 152). Measurement of MCL was made on 78% of attempts (n = 371), of BPD on 73% of attempts, and of both bones on 62% of attempts. The equation developed from BPD (mean absolute error (MAE) = 3.2 days) was similar to that developed from measurement of MCL (MAE = 3.3 days) in its capacity to predict stage of pregnancy. Accuracy of prediction was improved using equations that included mean values within litters for BPD (MAE = 2.5 days) and MCL (MAE = 2.6 days). Further improvement in predictive capacity was achieved using multiple regression equations developed from measurement of both bones (individual fetuses: MAE = 2.6 days; equations including mean values within litters: MAE = 2.2 days). The results demonstrate that ultrasound can be used to estimate stage of pregnancy in prolific ewes, and that the use of mean values for bone measurements from different fetuses within litters and/or measurement of bones with different growth allometry can increase the reliability of estimates. The utility of the procedure depends on the number of fetuses measured per ewe, the number of bones measured per fetus and, hence, the time required to measure bones and the degree of accuracy required.

Smad-interacting protein 1 is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic differentiation of C2C12 cells.

Up-regulation of liver/bone/kidney alkaline phosphatase (LBK-ALP) has been associated with the onset of osteogenesis in vitro. Its transcription can be up-regulated by bone morphogenetic proteins (BMPs), constitutively active forms of their cognate receptors, or appropriate Smads. The promoter of LBK-ALP has been characterized partially, but not much is known about its transcriptional modulation by BMPs. A few Smad-interacting transcriptional factors have been isolated to date. One of them, Smad-interacting protein 1 (SIP1), belongs to the family of two-handed zinc finger proteins binding to E2-box sequences present, among others, in the promoter of mouse LBK-ALP. In the present study we investigated whether SIP1 could be a candidate regulator of LBK-ALP transcription in C2C12 cells. We demonstrate that SIP1 can repress LBK-ALP promoter activity induced by constitutively active Alk2-Smad1/Smad5 and that this repression depends on the binding of SIP1 to the CACCT/CACCTG cluster present in this promoter. Interestingly, SIP1 and alkaline phosphatase expression domains in developing mouse limb are mutually exclusive, suggesting the possibility that SIP1 could also be involved in the transcriptional regulation of LBK-ALP in vivo. Taken together, these results offer an intriguing possibility that ALP up-regulation at the onset of BMP-induced osteogenesis could involve Smad/SIP1 interactions, resulting in the derepression of that gene.

Growth in width of the metacarpals--an investigation in human fetuses.

The process of growth in width of the human hand during fetal life has never been described. Do metacarpals grow concentrically and separation between the bones occurs through expansion of soft tissues? Or is growth eccentric, a process termed drift by Enlow, a relocation in space of organs? Hands of 10 spontaneously aborted fetuses (age range: between 14.5 and 24 weeks of gestation) were examined paying special attention to the bone bark. A thicker bone bark was taken as an indication of growth in that direction. The thickness of the bone bark was measured at the radial and ulnar sides at the level of the proximal and of the distal physes of the second to fifth metacarpals. A ratio of radial over ulnar bone bark thickness (R/U ratio) was calculated. The third metacarpal grew almost concentrically (R/U ratio 1.12 +/- 0.06). The second metacarpal grew in a radial direction (R/U ratio 3.29 +/- 0.19) and the fourth and more so the fifth metacarpal grew in an ulnar direction (R/U ratio 0.70 +/- 0.04 and 0.42 +/- 0.02, respectively). The differences in R/U ratios between every metacarpal were statistically significant for all comparisons P < or = 0.001. Fetal growth in width of the human metacarpals is eccentric and not concentric. It is concluded that during growth in width the metacarpals move away from the midline of the hand and that growth occurs through eccentric bone apposition rather than through soft tissue expansion.

The prenatal cranial base complex and hand in Turner syndrome.

From early childhood, Turner syndrome patients have a flattened cranial base, maxillary retrognathism, and short hands. There are, however, no studies that show when these genotype-determined abnormalities occur prenatally. The purpose of the present study was to measure craniofacial profile and hand radiographs of second trimester foetuses with Turner syndrome and compare the results with similar measurements from normal foetuses. The subjects consisted of 12 Turner syndrome foetuses, gestational age (GA) varying between 15 and 24 weeks, and crown-rump length (CRL) between 108 and 220 mm. The mid-sagittal block of each cranium was analysed as part of the requested brain analysis (pituitary gland analysis). This block and the right hand from seven foetuses were radiographed, and the skeletal maturity of the cranial base complex, i.e. the cranial base and the maxilla, was evaluated from the profile radiographs. Shape and size measurements in the cranial base were performed, and compared with normal values according to cranial maturity and to CRL. The cranial base angle in Turner syndrome was greater and the maxillary prognathism was reduced compared with the normal group. The dimensions in the cranial complex and in the hand showed that the bone lengths and distances in relation to CRL were generally smaller compared with normal foetuses. This investigation showed that the abnormal shape of the cranial base complex and the short hands in Turner syndrome are present prenatally.

An ultrasound-guided procedure to administer a label of DNA synthesis into fetal sheep.

A novel technique was developed to deliver a bolus dose of a DNA label into the peritoneal cavity of fetal sheep at 85-130 days gestation. Use of markers to identify the site of injection in fetuses from litters up to quadruplets, and immunohistochemistry to detect the DNA label, 5-bromo-2'-deoxyuridine (BrdU), confirmed the procedure was successful in 85% of cases. Duration of the procedure was (mean +/- SD) 44 +/- 16 min, and recovery from anaesthesia was rapid and uneventful in all cases. Fetal weight was estimated with a high degree of accuracy (residual standard deviation (RSD) = 297 g and r2 = 0.93, P<0.001) and the dose of label administered (110 +/- 33 mg BrdU/kg fetal weight) was adequate in all cases. BrdU detected in fetal nuclei following injection into amniotic fluid highlights the need for positive identification of the injection site in timed, short-term studies, and suggests potential to further develop the technique to investigate cellular events in fetal sheep younger than 85 days of gestation. The results demonstrate that the procedure can be used to determine in vivo whether or not nuclei have entered the S-phase of the cell cycle.

Ovariectomy and orchidectomy induce a transient increase in the osteoclastogenic potential of bone marrow cells in the mouse.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different timepoints after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Metacarpal growth and systemic markers of bone metabolism in the ovine fetus.

Bone metabolism was assessed in 56 Ile de Francex(RomanovxLimousine) fetal lambs killed between Day 80 and Day 145 of gestation. In each fetus, the length, width and weight, as well as the calcium and phosphorus content of the left diaphyseal metacarpal bone were measured. Plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities, bone ALP (B-ALP), osteocalcin (OC) and insulin-like growth factor-I (IGF-I) were assayed in these fetuses and in six newborn lambs from birth until one month after birth. Fetal growth is characterized by an increase in bodyweight, bone size and bone mineral content from Day 80 to Day 132 of gestation. These parameters did not significantly vary until birth. Plasma concentrations of IGF-1, OC and B-ALP increased from Day 80 to Day 132. Between Day 132 and birth, plasma IGF-I and B-ALP concentrations did not significantly vary, whereas plasma OC concentration decreased, confirming the usefulness of OC as a marker of osteoblastic activity and bone formation in the ovine species. The increase in plasma IGF-I, B-ALP and OC concentrations observed during the first two weeks of postnatal life indicate an intense skeletal growth in these animals, which was confirmed by the bodyweight growth curve.

Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones.

Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

[Gibbous hand deformity--carpe bossu]. / Handrückenhöcker--Carpe bossu.

Fiolle (10) was the first to describe a bony protuberance of a carpometacarpal joint and named it carpe bossu. This anomalous anatomical condition occasionally becomes a--widely unknown--common clinical entity. The reasons are discussed. The clinical diagnosis can be confirmed by a lateral tangential radiograph. Discomfort and pain are rare and can be treated usually by conservative means, surgery is seldom indicated.

Relative growth of the human metacarpals in the prenatal period: anatomic basis of preventive surgery for congenital deformities of the hand.

Twenty right metacarpal bones of Brazilian fetuses (ranging from 17 to 35 weeks post-conception) were studied by the allometric method to define the pattern of metacarpal growth. The metacarpal measurements analysed were their maximum length (ML), ossified shaft length (OSL) and midshaft breadth (MSB). These data were related to crown-rump length and the metacarpal weight to fetal weight. The increase in metacarpal measurements showed an allometric positive coefficient (kappa), highest in the OSL. The ML growth followed an axo-marginal pattern. The 3rd metacarpal showed the highest MSB kappa coefficient. The metacarpal weight showed the highest correlation coefficient (r) to the fetal data. These findings are important to the knowledge of human hand growth and are the basis of preventive surgery for congenital deformities of the hand.

Supernumerary epiphyses in the macaque (Macaca nemestrina).

The frequency, age distribution and development of supernumerary epiphyses in the hand and foot were radiographically documented in 98 macaque fetuses and infants. Extra epiphyses were observed only on distal metacarpal 1 and metatarsal 1, and were present in 15% of a cross-sectional sample and 20% of a longitudinal sample. They appeared coincidentally with the ossification of other secondary epiphyses of the hand and foot. Development of extra epiphyses in this species seems to be a normal phenomenon.

Growth inhibition of human embryonic and fetal rat bones in organ culture by rubella virus.

Paired organ cultures of metacarpal, metatarsal, and long bones of previable human embryos of 7 to 12 weeks' gestation and tibias of 17-day rat fetuses with inoculated with live or ultraviolet-inactivated rubella virus or control fluids and the growth of the bones was measured by increase in wet weight. In several cultures the ability of the human bones to incorporate 35S, a measure of rate of mucopolysaccharide synthesis, was tested. Growth of human and rat bones was retarded in cultures inoculated with live virus but not in cultures inoculated with inactivated virus or control fluids. Mean 35S uptake was increased by approximately 25% in virus-inoculated cultures of bones of 9- to 12-week human embryos. No histological abnormalities were seen. These findings suggest that (1) defective bone growth in congenital rubella is a direct effect of viral infection of bone, (2) a disorder of mucopolysaccharide syntheses may contribute to the osseous lesions that occur in this disease, and (3) organ cultures of human embryonic and fetal rat bones may serve as convenient models for studying the pathogenesis of this virus-induced congenital osteopathy.

A descriptive study of the rate of elongation and differentiation of the skeleton of the developing chick wing.

Differentiation of the wing skeleton is clearly visible in whole mounts from stage 24. It proceeds in proximo-distal and postero-anterior sequence. It is possible to map the part of the limb giving rise to each skeletal element as it appears and subsequently. This enables one to estimate: (a) a rate of elongation curve for each segment level, (b) the intrinsic rate of change of elongation of the cartilage, (c) a definitive extrapolation back to classical presumptive fate maps, (d) the normal range of variation in the proportion of the limb occupied by the three main segmental levels (stylopod, autopod, zeugopod).

Early prenatal attainment of adult metacarpal-phalangeal rankings and proportions.

As shown in 56 human embryos and fetuses between 15 and 104 mm in crown-rump length, "adult" metacarpal-phalangeal length rankings are attained by the seventh intrauterine week and near-adult bone-to-bone ratios or proportions by the theirteenth week. Micrometric measurements of optically-projected histological hand sections show relative elongation of the distals between the 15-29 mm and 30-44 mm crown-rump range, and relative reduction to radiogrammetrically-determined adult proportions by the 90-104 mm CRL.

Analysis of limb anomalies induced in vitro by vitamin A (retinol) in mice.

Forelimb buds of day 11 ICR-JCL mouse embryos were cultured on liquid medium consisting of 90% Eagle's MEM and 10% fetal calf serum. Experimental medium contained 10 iu/ml vitamin A alcohol (retinol). In controls four of five metacarpals chondrified and the epidermis began to keratinize after 3-4 days of culturing. In experimental explants many pycnotic cells were observed in the peripheral mesenchyme in the hand plate, especially in pre- and postaxial regions. Only 2-3 metacarpals chondrified, and keratinization was inhibited in the experimental explants. Uptake of labeled sulfate was suppresssed by vitamin A. Excess vitamin A is thus thought (1) to act directly on limb buds, and thereby to induce limb anomalies, (2) to induce cell death in the mesenchyme, (3) to suppres the formation of chondroitin sulfate, and (4) to inhibit keratinization.