Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

Summary Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20–30 days. Cells were cultured in either -minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 g/ml ascorbic acid, or the above medium supplemented with either 10 mM Na--glycerophosphate, 10^-8 M dexamethasone, or a combination of both. Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Glaprotein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction. Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both -glycerophosphate and dexamethasone. Cells associated with the nodules exhibited alkaline phosphatase activity. The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Glaprotein were present. X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite. The nodules were also examined for bone morphogenetic protein-like activity. Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats. Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers. This finding provided further support for the bone-like nature of the nodules. The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both -glycerophosphate and, particularly, dexamethasone.     

Immunocytochemical studies on the LHRH system of the Japanese quail: influence by photoperiod and aspects of sexual differentiation

Summary Immunocytochemistry was used to determine if photoperiod and/or sex have any effect on the pattern of the luteinizing hormone-releasing hormone (LHRH) system in the brain of the Japanese quail. Immunopositive perikarya were found within three major areas of the brain: the rostral paraolfactory lobe, the preoptic, and the septal region. A quantitative analysis of LHRH cell numbers was performed on male and female quail after two photoperiodic treatments: sexually mature birds exposed to 24 weeks of 20 h light: 4 h darkness (20L4D), and birds with a regressed reproductive system (induced by transfer from a photoregime of 20L4D to 25 short days of 8L16D). Two-way analysis of variance showed that short-day males display significantly (p < 0.05) more immunopositive perikarya (607 + 134) than long-day males (291 + 114), short-day females (293 + 103) or long-day females (330 + 92). The density of LHRH-immunoreactive nerve fibres and the intensity of the immunostaining in the median eminence were always greater in long-day sexually mature quail (male and female) than in animals exposed to 25 days of 8L16D. These results demonstrate that the LHRH system of the quail is influenced by photoperiod and mirrors sexual differentiation.     

Transformation with SV40 virus prevents retinoic acid inhibition of plasma membrane NADH diferric transferrin reductase in rat liver cells

Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40°C. When the cells are in the transformed state (grown at the permissive 33°C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.     

Biochemical differentiation in the tobacco flower probed with monoclonal antibodies

We have isolated a series of monoclonal antibodies that react to antigens in flowers of Nicotiana tabacum L. (tobacco) displaying specificity or preferentiality in their cell and tissue distributions. We immunized mice with extracts from tobacco flowers and then screened the hybridomas by enzyme-linked immunosorbent assay (ELISA) against extracts from leaves, sepals, petals, stamens and pistils; twenty five were chosen from the total screened. The antigens detected by about half of the antibodies were periodate-sensitive, implying that the epitopes were carbohydrate. Competition ELISA assays were used to determine if any antibodies were reacting to the same epitopes. Western blot analysis showed that while some antibodies reacted to specific bands, the bulk either failed to react or reacted to multiple bands, consistent with a glyco-conjugate nature for many of the antigens. Analysis of the spatial pattern of antigen distribution within tobacco flowers by immunolocalization showed that some antibodies recognized epitopes that were limited to very specific cells and tissues. We used the immunolocalization technique to analyze a mutant with stigmoid anthers: an antibody recognizing a pistil transmitting-tract antigen also reacted to cells in stigmoid anthers. Our results with this antibody set imply that biochemical differentiation within the tobacco flower includes cell-and tissue-specific glyco-moeities, and also that similarities, at the biochemical level, exist between a normal floral organ and the abnormal organ in a phenotype with a developmental switch.Abbreviations ELISA enzyme-linked immunosorbent assay - Fg immunoglobulin - kDa kilodalton     

Natural selection of allozyme polymorphisms: a microgeographic climatic differentiation in wild emmer wheat (Triticum dicoccoides)

Summary Allozymic variation in proteins encoded by 48 loci was analyzed electrophoretically in 1984 and 1985 in 137 individual plants of wild emmer wheat, Triticum dicoccoides, from a microsite in Yehudiyya, northeast of the Lake of Galilee, Israel. The test involved two climatic microniches in the open Tabor oak forest (1) sunny between trees and (2) shady under trees' canopies. Significant genetic differentiation at single-, two- and multilocus structures was found between neighboring climatic niches, which were only separated by a few meters. Our results suggest that allozyme polymorphisms in wild emmer wheat are partly adaptive, and differentiate primarily at the multilocus level by climatic factors presumably related to aridity stress.     

Somatotropin antagonism of insulin-stimulated glucose utilization in 3T3-L1 adipocytes

It is well established that somatotropin (GH) antagonizes insulin action in vivo and that supraphysiologic concentrations of GH frequently result in insulin resistance and glucose intolerance. However, the demonstration of an anti-insulin activity by GH in vitro has been difficult. This study, therefore, set out to determine whether cultures of 3T3-L1 adipocytes could be used to examine the anti-insulin activity of GH. The ability of insulin to stimulate glucose utilization by 3T3-L1 adipocytes increases approximately five-fold during the first 4 days following treatment of the cells with a differentiation medium. It was found that glucose utilization in 3T3-L1 adipocytes is regulated in a reciprocal fashion by insulin and GH. Bovine or human GH directly inhibit up to 50% of insulin-stimulated [14C]-glucose incorporation into lipids in a concentration-dependent manner. The 3T3-L1 sensitivity to GH appears to be at the maximum (50% inhibition of an insulin response) immediately following removal of the cells from the differentiation medium and remains essentially constant during the subsequent 4 days. The GH inhibition of insulin action does not appear to be due GH enhancement of cellular degradation of insulin, competitive binding of GH to the insulin receptor, or GH-induced decrease in cell number. The 3T3-L1 adipocyte system appears to be a sensitive and reliable in vitro model with which to study the molecular mechanisms involved in both GH antagonism of insulin action and development of hormone responsiveness during cellular differentiation into adipocytes.     

Extramacrochaetae,a trans-acting gene of the achaete-scute complex of Drosophila involved in cell communication

Summary Phenotypic analyses of genetic combinations involving the gene extramacrochaetae (emc) reveal its participation in the differentiation of both sensory elements and wing veins. The study of near-amorphic alleles of emc in mitotitc recombination clones indicates that it also affects cell proliferation. These clones show abnormal sizes, shapes and spatial distribution. They differentiate extra sensory elements as well as extra veins. A gain of function mutation in the gene causes opposite phenotypes in both differentiation systems. The effects of the mutant on proliferation and patterning are consistent with the emc gene being involved in the transfer of information between neighbouring cells, which leads to the spatial expression of the achaetescute gene complex and genes involved in vein formation.     

TPA对原代白血病细胞的诱导分化作用

本文报告了TPA对32例不同类型白血病细胞的体外分化诱导结果。TPA(1.6×10~-7M)可诱导急性非淋巴细胞(ANLL)白血病细胞迅速出现单核巨噬细胞分化标志:细胞贴壁、胞浆丝状伪足形成,具有类似巨噬细胞的形态改变及相应的细胞化学反应特征。急性淋巴细胞白血病(ALL)和桨细胞白血病(PCL)细胞不发生上述变化,表现为细胞聚集成闭现象。慢性淋巴细胞白血病(CLL)出现桨细胞样形态转化。初发与复发病例的诱导反应相类似。TPA体外诱导分化实验,有助于了解病人白血病细胞的分化潜能,对于鉴别粒单系和淋巴系两类白血病,尤其对于用常规方法分型困难的低分化白血病有一定的临床诊断意义。