Pathogenesis of radiation-induced abnormalities of the bones and joints of white rat embryos

After X-radiation of pregnant rats on the 10th day of pregnancy, in 50% of the fetuses studied subtotal aplasia of the tibial bone anlage and decreasing number of the metatarsus and finger phalanges anlages are observed. Radiation on the 11th day of embryogenesis does not result in anomaly formation of the thoracic and pelvic extremities. After radiation on the 12th day of embryogenesis, the most specific anomaly of the pelvic extremity is phocomelia. The thoracic extremity skeleton lesions are revealed as an ulnar type of distal ectromelia, or axial ectromelia. After radiation on the 13th--14th day, hypoplasia of the bone anlages, that make zeugopodium, autopodium, is observed. After radiation on the 13th day, a partial or total aplasia of the fibular bone anlage can take place. In all the fetuses a sharp decrease in number of the hand and foot bone anlages is observed; it is connected with a total aplasia of some of them and with fusion of the others. A specific feature for radiation lesions of the extremity skeleton is that the oppositely situated anlages of the bones do not separate from each other. This results from certain disturbances in the joint interzone formation at early stages of embryogenesis and from underdevelopment of the joint cleft. Qualitatively different radiation anomalies of the extremity skeleton development are formed as consequence of disturbances in morphogenetic processes of determination: migration, proliferation, morphogenetic cell death and differentiation.     

Ossification of the cartilage skeleton of the extremities of human embryos

Certain sequence in appearance of ossification points has been stated in the cartilage models of the superior and inferior extremities of the human embryos at the end of the embryonal and the beginning of the fetal periods of development. The change in the size (length) of the ossification points in anlages of the long tubular bones during the successive stages of embryogenesis is of linear character and can be described by means of the equation y = ax + b, where y--age of the embryo (days), x--length of the osseous points. Coefficients a and b are calculated for estimation the age of the embryos according to the length of the osseous points in the anlages of the brachial, femoral and radial bones.     

Differentiation of joints in transplants developing on chorioallantois

The wing of the chick embryos (the 17th-21st stages of development according to Hamburger--Hamilton) were transplanted on the chorioallantois of the chick embryo-recipients, incubated for 8.5-9.5 days. Differentiation of the joints was studied in serial histological sections and in translucent preparations of the skeleton stained with alcian blue. The transplants for the investigation were taken on the 1st-11th days after transplantation. In the transplants all three segments of the wing always developed. The development of the external form of the extremity, chondrogenesis and osteogenesis of the skeletal anlages were about 24 h late. Histological changes, specific for the early period of the articular interzone and cleft formation corresponded to the control embryos data, but were one day younger. In future the changes did not progress, and passed into regression, demonstrating as fusion of the articular surfaces. In the transplants blood vessels formed networks of irregular form that surrounded the articular zones. Some branches run from them into mesenchyme, situating around the joint. According to the literature data, these vessels are connected with formation of the articular cleft and in the control embryos blood vessels of the articular capsule develop from them. In the transplants they are dilated, twisted (especially in the ulnar joint area) and do not penetrate into the developing prechondral and then into the cartilage bridges of the fusing articular surfaces. Numerous blood accumulations, as well as extravasates are often seen near the deformed anlages of bones. Thus, disturbance of blood supply in the transplants and lack of innervation in them, discussed in the literature, result in fusion of the articular surfaces.     

Glycosaminoglycans at the early stages of osteogenesis of the human long tubular bones

In anlages of long tubular bones of 68 human embryos and prefetuses (6-12 weeks of the prenatal development) changes in content and composition of glycosaminoglycans have been studied at early stages of osteogenesis. Histochemical reactions with brown basic, alcian blue, using the method of critical concentration of electrolyte and toluidine blue have been applied at various low values of pH with corresponding control experiments. For ultrastructural investigation of glycosaminoglycans localization the electron histochemical reaction with nitrogen acidic bismuth has been used. The most active synthesis of glycosaminoglycane is observed in chondrocytes of the proliferative zone of the epiphyseal cartilage of the anlages. In chondrocytes of the columnar zones the synthesis of glycosaminoglycans subsides, however, the mass of the substances increases on the surface of cytolemma of chondrocytes and in the intercellular matrix. During the process of development of the human long tubular bone analages contents of low sulfated glycosaminoglycans decrease with a simultaneous increase of concentration of these components, having a more acidic reaction. An essential role of polysaccharides and their complexes with proteins in ossification and mineralization of the human long tubular bone anlages is supposed.     

Alkaline phosphatase and 5'-nucleotidase in early osteogenesis in man

By means of histochemical techniques at light and electron microscopic levels, as well as immunomorphological, biochemical and immunochemical methods localization and dynamics of alkaline phosphatase and 5'-nucleotidase contents have been determined in anlages of long tubular bones in 85 human embryos and prefetuses from the 6th up to 12th week of the intrauterine development, obtained as a result of artificial abortions in healthy women. The greatest activity of the enzymes studied is revealed in areas of an intensive osteogenesis and mineralization. Also, by means of the immunofluorescent method alkaline phosphatase of a placental type is revealed, that is not revealed, however, immunochemically. With increasing time of the intrauterine development, thermostability of alkaline phosphatase increases.     

Novel method for the establishment of cardiomyocytes derived from rat embryonic stem cells in vitro

Cardiomyocytes were differentiated from embryonic stem cells (ES cells) derived from spontaneous dwarf rats (SDR) in vitro. The two-cell stage embryos were cultured in alpha-MEM supplemented with 10% fetal calf serum and embryotrophic factors (ETF). ETF were isolated from the conditioned medium of the SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. When two-cell stage rat embryos developed into tri-laminal germ disc embryos (flat type), colonies composed of small round cells were isolated by the colonial isolation method and used to establish an ES cell line. The ES cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum and 1 ng/mL of leukemia inhibitory factor. Embryoid bodies were made by the hanging-drop method using 1 x 10(7) ES cells/mL. The embryoid bodies differentiated and grew to form an embryonic monster in ETF-supplemented medium using Rose's circumfusion apparatus for about 1 month. The anlages of beating hearts in embryonic monsters were collected using a glass capillary. The anlages were cut into small pieces using razor blades and dissociated with trypsin-EDTA/PBS(-) solution. The resultant single cells were cultured in growth medium and used to establish a myocardial cell line. The cell line was subcultured for more than 25 passages and confirmed as showing the morphological and ultrastructural characteristics of cardiomyocytes.     

Substrate basis of the adhesion of the adenohypophyseal and diencephalic cell layers and cell anlagen in chick embryos]

At the 16-18th stages of development of the chicken embryos adhesion of cell layers of the adenohypophysis and diencephalon anlages is provided with a submicroscopically regulated complex of neutral and acid polysaccharides, bivalent kations and probably proteins. The mechanisms of cells contact in each of the layers with this complex are different.     

Lansing man: a half century later

One of the major fossil man finds from the Plains Area of the United States and one of the few from Kansas are the “Lansing Man” skeletons. The discovery was in February 1902 on the west bank of the Missouri River, south of Leavenworth near the town of Lansing, Kansas. Much was written about this skeleton following its discovery and Ale? Hrdli?ka's only trip to Kansas in October 1902 was to observe the skeletons and the site of its discovery. Numerous articles were written suggesting that “Lansing Man” was many thousands of years old. Geologists could not agree on an age of the skeletons, an adult male and a six to seven year old child, because they were discovered in deposits of slumped loess, confusing the geological picture. Hrdli?ka states that the skeletons were physically identical to Indians of that region at the period of historical contact. William Holmes had the skull sent to the U. S. National Museum and the remaining bones were placed in the Museum of Natural History at the University of Kansas. While on the staff of the University of Kansas, I had Carbon-14 tests conducted on bones from the lower limbs by three separate laboratories. These dates range from 2660 to 5020 B.C. with an average date of four samples (1 each from Geochron Laboratories and the University of Michigan and two from the Smithsonian Institution) of 3579 B.C. This suggests that the “Lansing Man” skeletons are Early Middle Archaic and not Paleoindian. They do, however, represent the oldest known human skeletons from Kansas.     

The development of the clavicle in man

The development of the human clavicle was studied in 50 to 60 d old human embryos. Our findings are summarized as follows: The whole clavicle develops from a cartilaginous anlage. In the middle part of the clavicle, an osseus cuff develops very early by the ossification in the perichondrium. In the lumen of this cuff, a cartilaginous cork persists which is resorbed and replaced by bone and marrow later than in other bones. It is possible that cartilaginous nests may persist in the middle part of the clavicle. In both extremities of the clavicle, the normal enchondral ossification exists as it is described in other anlages. It is difficult to explicate the syndrome of the cleido-facial and cleido-cranial dysostoses only as disturbances of the endesmal ossification.     

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.     

Disorders of vaginal development in the progeny of white rats exposed to x-ray irradiation

One hundred and twenty white rat embryos 13-22-day-old have been irradiated with x-rays (the dose 250 R) on the 12th-14th day of embryogenesis. The embryos have been divided into series of sagittal, frontal, transversal sections and stained by means of general histological methods. The irradiation performed on the days mentioned does not affect formation of the paramesonephric ducts. In all the experimental animals the caudal end of the paramesonephric duct is separated from the mesonephric duct as a solid cellular cord in which the lumen appears later. In the experimental females the disturbances developed after irradiation are manifested first of all in retardation of the main stages of the organ's formation; the retardation is observed: in fusion of the paramesonephric ducts, in resorption of the medial septum between the fused ducts, in separating the sinuous part of the vagina from the urogenital sinus, in recanalization of the vaginal epithelial cord. More severe lesions are presented as agenesia of the vaginal sinuous part and as its atresia represented by a transversal septum of the organ. The disorders in the vagina development are depended on massive primary necrobiotic radial lesions of the mesenchymal cells around the epithelial anlages of the small pelvis.     

Phylogenetic analysis of ancient mitochondrial DNA lineages of human remains found in Yakutia

Molecular genetic analysis of ancient human remains is mostly based on mtDNA owing to its better preservation in human bones in comparison with nuclear DNA. A study was made of mtDNA extracted from human skeletons found in graves in Yakutia, in order to determine the haplotypes and to compare them with lineages of modern populations. Ancient DNA was extracted from fragments of three skeletons of Yakut graves at At-Dabaan, Ojuluun, and Jaraama sites (dating back to the 18th century) and two skeletons of the Late Neolithic Kerdugen grave (2000–1000 B.C.). All graves were found in central Yakutia (Churapchinskii, Khangalasskii, and Megino-Khangalasskii districts of Yakutia). Five different haplotypes belonging to specific Asian haplogroups were identified. The mtDNA lineages of Yakut graves belong to haplogroups C4a, D5a2, and B5b. The results indicate the continuity of mitochondrial lineages in the Yakut gene pool in the past 300 years. The haplotypes of two humans from the Kerdugen site graves belong to haplogroups A4 and G2a/D. These haplotypes were compared with those of 40000 Eurasian individuals, including 900 from Yakutia. No exact matches were found in Paleo-Asian populations of Chukchi, Eskimos, Koryaks, and Itelmen. Phylogenetically close haplotypes (±1 mutation) were found in Yakut and Evenk populations, as well as in some populations of China and South and West Siberia.     

Nucleocytoplasmic traffic of proteins.

We have used the synchronized formation of a mixed cytoplasm upon heterokaryon formation as a model for investigating the cisternal-specific transport of resident proteins between neighboring Golgi apparatus. Rat NRK and hamster 15B cells were fused by UV-inactivated Sindbis virus and then incubated for various time periods in the presence of cycloheximide. The resident Golgi apparatus proteins, rat GIMPc and Golgp 125, were localized with species-specific monoclonal antibodies. Immunofluorescent colocalization of rat and hamster Golgi membrane proteins was observed with a t1/2 of 1.75 h at 37 degrees C. Colocalization of resident, but not transient, Golgi membrane protein was concomitant with formation of a large extended Golgi complex and was accompanied by the acquisition of endoglycosidase H resistance by preexisting Golgp 125. Dispersal of the extended Golgi complex by nocodazole revealed that colocalization of resident Golgi proteins was due to intermixing of proteins in the same Golgi element rather than overlapping of closely apposed Golgi structures. Incubation of the polykaryons at 20 degrees C inhibited both the colocalization of GIMPc and Golgp 125 and the formation of an extended Golgi complex. Little change in the number of cisternae/stack in cross sections of the Golgi apparatus was observed upon cell fusion, and in the extended Golgi complex the hamster resident protein remained localized to one side of the Golgi stack. Surprisingly, the morphological identity of the rat and hamster Golgi units appeared to be maintained in the heterokaryons. These results suggest that the intermixing of resident Golgi membrane proteins requires direct physical continuity between Golgi elements and that resident Golgi membrane proteins are preferentially excluded from the non-clathrin-coated transport vesicles budding from Golgi cisternae.     

Action of acetazolamide on the chick embryo during late development.

Acetazolamide was injected into chick embryos on the 14th or 15th day of incubation. Doses ranging between 5 and 10 mg per egg produced a retardation in the growth of long bones. The affected bones contained a normal proportion of mineral as determined by ashing and presented a normal histological picture. On the basis of these findings, it is suggested that the alterations were not due to a specific direct effect of the drug on bones. The incorporation of 131-I by the thyroid glands of acetazolamide-injected embryos was analyzed radioautographically and quantitated on the same 6 mu-paraffin sections, with a thin window Geiger counter. The incorporation appeared notably reduced 3 h after the injection of acetazolamide and the reduction persisted for a least 24 h.the electron microscopical observation of thyroid follicular cells from similarly treated embryos showed that the cytological characteristics indicating an active protein synthesis were unmodified with respect to those found in control embryos. These results may indicate that acetazolamide inhibits the iodination of the throid hormone without interfering with the synthesis of the globulin. It is suggested that the growth retardation observed in the embryos treated with acetazolamide may be secondary to the action of the drug on the thyroid gland, although this action appears to be a transitory one.     

Possible mechanisms of cadmium fetotoxicity in golden hamsters and mice: uptake by the embryo, placenta and ovary.

Pregnant golden hamsters and mice of different gestational ages were injected intravenously with 109CdCl(2). The whole animal or the uterus and embryos were submitted to autoradiography. Cadmium administered on the 8th day accumulated in the primitive gut of the embryos. No cadmium was detected in the embryos after administration on or after the 9th day (hamster) and 11th day (mouse). This finding can be explained by the ability of cadmium to pass from the yolk-sac cavity into the primitive gut (where it is absorbed) before the closure of the vitelline duct but not later. This uptake by the embryo might explain the severe malformations produced by cadmium given on the 8th day as compared with the 9th day in the hamster. Cadmium is also heavily accumulated in the decidua (mainly the antimesometrial part), the yolk sac, the ectoplacental cone, and later in the chorioallantoic placenta-possibly disturbing the maternal-embryonic relationship and fetal nutrition. A high accumulation in the CL and the follicles and in the pituitary may also disturb reproductive function.     

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.     

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.     

The density of long limb bones and the percentage ash weight of the skeleton of Macaca mulatta

The gravimetric density of humeri, radii, femora and tibiae from a series of 274 male and female skeletons of rhesus monkeys, Macaca mulatta, was determined for fetal, young and adult periods. The ages of 171 of the animals were known: they ranged from 57 days of gestation to 13.6 years; the ages of an additional 103 skeletons were estimated. The mean density of the fetal bones was found to increase linearly with age and was higher for males than females, and higher for the superior than for the inferior limb bones. During the young period the pattern of increase in density can be represented by a power-type curve, and the density is significantly higher in females than in males and in superior than in inferior limb bones. The densities of the long limb bones of the adult skeletons show a slight, but not significant, negative trend with increasing age. In this age group the mean densities are higher for males than females and higher for the superior than for the inferior limb bones. The percentage ash weight was determined for the total skeleton and for 21 subdivisions of 23 postnatal skeletons with estimated ages. The skull and long limb bones were found to have higher mean percentage ash weights than the vertebral segments and the sternum. Both the density and the percentage ash weight of the Macaca mulatta skeletons examined exceed those found in our earlier studies of the human skeleton.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.