Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.     

The establishment of vascular-derived microenvironments in the developing chick wing

The results of previous studies on the temporal sequence of limb vascularization suggest that the prospective myogenic and chondrogenic areas of the mesoderm are distinguished by a differential vascularization pattern prior to the overt expression of muscle- and cartilage-specific phenotypes. The experiments presented here are designed to reveal the dynamic aspects of vascular flow in the limb by the observation of how an inert, particular tracer (india ink) is mobilized and dispersed at specific points in the mesoderm. Data are presented as a temporal sequence of fluid flow "maps" which detail both the rate and the direction of vascular flow in the limb. It is proposed that not only does the vasculature compartmentalize the mesoderm into prospective myogenic and chondrogenic zones but also that these broad areas are subcompartmentalized into discrete microenvironments that are spatially distinct with regard to their capacity for transporting the carbon particles. The developmental significance of this observation may be that limb mesodermal cells are granted precise, "positional" information in the form of the specific nutrient and oxygen levels they encounter during critical, or decisional, phases of morphogenesis.     

Human bone marrow stromal cells express an osteoblastic phenotype in culture

Summary This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [³H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D₃ by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with⁴⁵Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.     

Transcriptional diversity in myogenesis.

Duodenal morphogenesis in the chick embryo has been studied to see if changes in organization of extracellular fibers are associated with villus formation. Three major processes occur during morphogenesis; longitudinal ridges form, then these ridges become zigzag in shape, and finally a double row of villi form from each zigzag ridge. Tissues of different developmental stages were progressively trypsinized to remove cellular material and were prepared for scanning microscopy to show the organization of extracellular fibers. Results show that fiber systems of increasing complexity form as the dudoenum develops, and suggest that some cellular events such as initial ridge formation precede these changes. Tissues with longitudinal ridges contain only randomly organized fibers. In tissues with zigzag ridges, oriented fibers appear along the ridges and some extend laterally from flexures of each zigzag ridge, but interridge fibers are still randomly organized. When villi form, fibers in the body of the villus are random but fibers at the base of villi and between villi are highly oriented. Transmission microscopy and collagenase treatment of partially trypsinized tissue indicate that most, if not all extracellular fibers viewed by scanning microscopy are collagenous. Therefore, since collagen fiber organization is so closely related to morphogenetic changes, we presume it plays an important role.     

The possible differentiation of osteogenic elements in vitro from chick limb mesodermal cells. I. Morphological evidence

In the developing chick limb bud, myogenic, fibrogenic, chondrogenic, and osteogenic tissues are derived from embryonic mesenchyme. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, fibrocytes, and myocytes can be identified on the basis of morphological and biochemical parameters. The studies reported here clearly demonstrate that, in similar cultures, crystalline calcium phosphate material is deposited in the chondrocytic and fibrocytic matrices. Such crystalline material is not observed before Day 10 of culture life. Subsequent to Day 10 of culture, first amorphous, then crystalline calcium phosphate is observed. On the basis of light and electron microscopic analysis, it appears that the in vitro calcification phenomenon closely resembles the morphological and temporal sequence of osteogenesis observed in vivo.     

Epidemiological Characteristics of Strongyloidiasis in Inhabitants of Indigenous Communities in Borneo Island,Malaysia

Epidemiological study on strongyloidiasis in humans is currently lacking in Malaysia. Thus, a cross-sectional study was carried out to determine the prevalence of Strongyloides stercoralis infection among the inhabitants of longhouse indigenous communities in Sarawak. A single stool and blood sample were collected from each participant and subjected to microscopy, serological and molecular techniques. Five species of intestinal parasites were identified by stool microscopy. None of the stool samples were positive for S. stercoralis. However, 11% of 236 serum samples were seropositive for strongyloidiasis. Further confirmation using molecular technique on stool samples of the seropositive individuals successfully amplified 5 samples, suggesting current active infections. The prevalence was significantly higher in adult males and tended to increase with age. S. stercoralis should no longer be neglected in any intestinal parasitic survey. Combination of more than 1 diagnostic technique is necessary to increase the likelihood of estimating the ‘true’ prevalence of S. stercoralis.     

Double-inhibitor and uncoupler-inhibitor titrations. 1. Analysis with a linear model of chemiosmotic energy coupling

The results of double-inhibitor and uncoupler-inhibitor titrations have been simulated and analyzed with a linear model of delocalized protonic coupling using linear nonequilibrium thermodynamics. A detailed analysis of the changes of the intermediate delta muH induced by different combinations of inhibitors of the proton pumps has been performed. It is shown that with linear flow-force relationships the published experimental results of uncoupler-inhibitor titrations are not necessarily inconsistent with, and those of double-inhibitor titrations are inconsistent with, a delocalized chemiosmotic model of energy coupling in the presence of a negligible leak. Also shown and discussed are how the results are affected by a nonnegligible leak and to what extent the shape of the titration curves can be used to discriminate between localized and delocalized mechanisms of energy coupling.     

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.