C-Met expression and mechanical activation of satellite cells on cultured muscle fibers.

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.     

Isoform-Specific Attachment of Osteoprogenitors to Laminins: Mapping to the Short Arms of Laminin-1

The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey (Lampetra planeri Bl.).

1. The biosynthesis of 18-19S thyroglobulin has been studied in a larval and adult freshwater lamprey (Lampetra planeri Bl.). 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates (125I, [3H]leucine and various [3H]carbohydrates). 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18-19S Tg), containing a small proportion of labeled T3 and T4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. In adults, the biosynthesis of 18-19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18-19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. Some 18-19S Tg is apparently stored in the endostyle.