Implanting mouse embryo stain with a LNF-I bearing fluorescent probe at their mural trophectodermal side.

Mouse embryos at implantation stage were stained successfully with lacto-N-fucopentaose I (LNF-I) bearing neoglycoprotein labeled with rhodamine synthesized by us for the first time. The fluorescent neoglycoproteins carrying LNF-II, -III, LND-I, or LNT failed to stain the embryos. The embryo was stained only at the cell surface of trophectoderm at the mural side. Since the attachment of the mouse embryo to the uteric epithelium occurs at its mural side trophectoderm and LNF-I is the key substance in mouse implantation (Lindenberg, S. et al, (1988) J. Reprod. Fert. 83, 149-158), the material stained with the probe carrying LNF-I appears to be the molecule responsive to attachment to the endometrium surface and leading to implantation.     

Interaction of amylose and other α-glucans with hydrophobic fluorescent probe (2-p-toluidinylnaphthalene-6-sulfonate)

Fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was enhanced in the presence of maltooligosaccharides, amylose, and other α-glucans. The dependence of relative TNS fluorescence intensity per glucose unit on chain length of oligosaccharides was examined. The values of binding constant and thermodynamic parameters, assuming the 1:1 complex for TNS-amylose (number-average degree of polymerization, DP_N = 17), were determined by the fluorescence titration. The values of thermodynamic parameters for 1:1 complex formation of TNS-α- and β-cyclodextrins were also determined and compared with those of TNS-amylose (DP_N = 17). The fluorescence intensity of TNS in the presence of amylose (DP_N = 600) decreased by the action of glucoamylase and taka-amylase A. The fluorescence of TNS-amylose (DP_N = 17) system increased with the increased ionic strength. In the presence of pullulan, TNS fluorescence was also enhanced and decreased by the action of pullulanase. Amylopectin enhanced TNS fluorescence rather more strongly than amylose (DP_N = 17) at the same concentration. In the presence of dextran, the fluorescence of TNS was scarcely enhanced. The degree of fluorescence enhancement of TNS in the presence of α-glucans seems to reflect the structures of α-glucans in solution, since TNS fluorescence is enhanced in the hydrophobic environment or by the disturbance of free intramolecular rotation.     

Interaction of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene with biomembranes

The relationship between the conditions of membrane labelling by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and its fluorescence parameters was investigated. In the labelling solutions prepared by the usual method, the presence of DPH microcrystals was revealed which led to the lower resultant fluorescence anisotropy values. Lower labelling efficiency was observed with DPH solutions in tetrahydrofuran when compared with solutions in acetone. Modifications of the labelling procedure are proposed which give better reproducibility of the results. There modified method involves the preparation of a 2 X 10(-4) mol. 1(-1) DPH stock solution in acetone, a 100-fold dilution in an appropriate buffer, subsequent bubbling through with nitrogen for 30 min and mixing the resulting solution with cell/membrane suspension in a 1:1 (v/v) ratio. Changes in intensity, anisotropy and spectra of DPH fluorescence in the course of membrane labelling were studied. A two-stage model of the incorporation of DPH into membranes was proposed, according to which DPH molecules first quickly adhere to the membrane surface and then are slowly translocated to the apolar regions of the membrane.     

Localization of tropomyosin in mouse embryo fibroblasts.

Antiserum to chick skeletal muscle tropomyosin was used to localize tropomyosin in mouse embryo fibroblasts by the indirect fluorescein labeled antibody technique. Specific staining was observed cytoplasmic fibers, which extended out into the cell processes. The staining pattern in these cells is similar to that previously described by others for actin. This observation suggests that in fibroblasts tropomyosin, like actin, is localized in fibers in the cytoplasm.     

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.     

A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution.

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.     

Interaction of the fluorescent probe diS-C3-(5) with lecithin-cholesterol membranes

Behaviour of fluorescent carbocyanine probe disS-C3(5) in the egg lecithin-cholesterol membrane suspension was studied in relation to the lecithin/cholesterol ratio. The partition coefficient of the probe between aqueous and lipid phases decreases unlinearly with increase of cholesterol molar part in a bilayer. This parameter over molar part units was estimated to be (2.4 +/- 0.1) X 10(6) for egg lecithin membranes and (1.8 +/- 0.2) X 10(6) for 10 mol% cholesterol, (1.2 +/- 0.1) X 10(6) for 20, (0.8 +/- 0.1) X 10(6) for 30, and (0.48 +/- 0.02) X 10(6) for 50 mol% cholesterol. It is suggested that the probe partition coefficient value consists of two components: one caused by pure lecithin bilayer regions and another by local lecithin concentration fluctuations in the mixed lecithin-cholesterol regions.     

Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe

Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

Response of plateau-phase mouse embryo fibroblasts to ultra-violet light.

Clonogenic survival was measured in plateau-phase cultures of the 10T1/2 mouse cell line exposed to 254 nm ultra-violet light. The survival curve was found to be biphasic, Do for the two components being 37 and 1191 erg/mm2 respectively. This extreme resistance at higher doses can only be partly accounted for by the increased cytoplasmic absorption of U.V.L. due to an increased thickness of plateau-phase cells. When the cultures were held for 24 hours in plateau phase in conditioned medium after irradiation, recovery yielding a 1.4-fold enhancement of survival was found at higher doses. This recovery process was inhibited by neither caffeine nor cycloheximide. When caffeine was given for 48 hours after sub-culture, the effect on survival was also negligible. We propose that this plateau-phase recovery process is associated with excision repair of DNA adducts induced by U.V.L. Delayed sub-culturing favours the excision mode of repair and renders the post-replication mode less necessary.     

S phase mouse embryo fibroblasts secrete varying amounts of a 45,000 dalton protein

Synchronized cultures of mouse embryo fibroblasts upon release from hydroxyurea (HOU) arrest, secreted several proteins of which a polypeptide of molecular weight 45,000 (45K) was barely visible in the conditioned medium of cells that synthesized DNA at peak levels. The quantity of the 45K protein was higher in the medium of HOU arrested cells and the level got progressively reduced as the cells entered into the DNA synthetic phase. Conditioned media containing the 45K protein inhibit DNA synthesis when added to synchronized cultures. These results suggest that the 45K secreted protein may be involved in the autocrine regulation of turning-off of DNA synthesis at the end of S phase.     

Interaction of spectrin with hydrophobic agaroses

Purified spectrin was found to interact strongly with hydrophobic agaroses such as Phenyl- or Octyl-Sepharose, in the presence of EDTA. From the complexes formed spectrin was eluted with ethylene glycol but not with low ionic strength solutions. The binding capacity of spectrin increased with increasing ionic strength of the equilibration buffer and showed but little dependence on its pH value. The fragments obtained by proteolysis of spectrin carried out under mild conditions were also found to bind strongly to phenyl-agarose, and were eluted with ethylene glycol. The fractions eluted with ethylene glycol contained two closely related polypeptides of Mr 65,000 and 60,000.     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Interaction of the voltage-sensing fluorescent probe diS-C3-(5) with dipalmitoylphosphatidylcholine liposomes

The interaction of the probe diS-C3-(5) with dipalmitoylphosphatidylcholine (DPPC) liposomes has been studied using fluorescence and differential scanning calorimetry (DSC). The partition coefficients (K) of the probe for the lipid and the aqueous phase (in terms of molar part units) were (1.20 +/- 0.4) X 10(6) at 45 degrees C and (0.50 +/- 0.07) X 10(6) at 23 and 36 degrees C. In terms of volume concentration units, these values correspond to Kp = (2.88 +/- 0.10) X 10(4) and Kp = (1.20 +/- 0.17) X 10(4), respectively. DSC thermograms were practically identical both for large unilamellar and multilamellar liposomes. The main transition peak remained practically unchanged over the entire range of the probe concentrations used. The pretransition could be observed up to maximal probe concentrations applied and it widened and shifted from 35.4 degrees C in pure DPPC to approximately 32 degrees C at a probe/lipid ratio of 0.027. These results suggest that in both quasicrystalline and liquid crystalline lipid bilayers the probe molecules are included in "defects" between structurally ordered microregions (microdomains or clusters). The dependence of the fluorescence response on the transmembrane potential in a suspension of unilamellar DPPC vesicles suggest that the equilibrium thermodynamic model is valid for liquid crystalline bilayers.     

Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism

Immunofluorescence with an antiactin antibody and electron microscopy were used to study the distribution of actin in cultured mouse fibroblasts during treatment with inhibitors of energy metabolism. The inhibitors induce gradual disorganization of actin-containing microfilament bundles. At the first stage of the process the bundles degrade into separate fragments; later only small patches of actin can be found in the inhibitor-treated cells. This transformation takes about 90 min and is fully reversible as microfilament bundles are recovered after incubation of the cells in the inhibitor-free growth medium. The inhibitors do not alter actin distribution in the presence of glucose. This shows that their action is due to a reduction of the ATP level in the cells. A 90 min incubation with the inhibitors does not markedly alter either the cell shape or the microtubule system. Inhibitors of the energy metabolism prevent cytochalasin action on cells. Cytochalasin B (CB) or cytochalasin D (CD) rapidly disorganize the microfilament bundles and cause cell arborization. However, microfilament bundle destruction in the cells incubated in the mixture of cytochalasin and any of the inhibitors requires 90 min and is not accompanied by dramatic changes in the cell morphology, so the process is indistinguishable from microfilament bundle destruction in the presence of the inhibitors alone.     

Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of platinum replicas

We have studied the distribution of cytoskeletal elements in detergent-extracted mouse embryo fibroblasts using the platinum replica technique. It was shown that lamelloplasm can be subdivided into three zones: 1) the ruffle edge with dense microfilament meshwork; 2) the sparse zone adjacent to the ruffle edge and containing relatively few cytoskeletal elements; 3) the lamella proper occupied with a three-dimensional network of microfilaments, microtubules, intermediate filaments; this zone contained adhesion plaques corresponding to cell-substrate focal contacts and associated with the microfilament bundle ends. The cytoskeleton structure of the central (endoplasm) region of the cell was markedly different from that of the lamelloplasm. Its main feature was a dense microfilament sheath at the dorsal cell surface. Sites of microfilament bundle convergence can be visualized near the nucleus after partial removal of the sheath by more complete detergent extraction.     

Secreted proteins of quiescent,serum-stimulated and over-confluent mouse embryo fibroblasts

Quiescent and proliferating cultures of Swiss mouse embryo fibroblasts were pulse labelled with [¹⁴C]-amino acids and the newly synthesized proteins that were secreted into the medium were resolved by electrophoresis on Polyacrylafde gradient gels. Conditioned media obtained from quiescent cultures that were stimulated to grow by the addition of 20% fetal calf serum showed the presence of two unique polypeptides of molecular weights 48000 and 26000. A polypeptide of molecular weight 45000 was present in increased amounts in serum-stimulated cells than in quiescent cells. This protein was also superinduced in quiescent cells by cycloheximide treatment. Mouse embryo fibroblasts grown under over-crowded conditions secreted two proteins of molecular weights 35000 and 11000. The 35 K polypeptide was shown to be related to the major excreted protein of transformed cells, since it was immunoprecipitated by an antiserum to major excreted protein. These results indicate that the 48 K and 26 K proteins may be proliferation specific proteins, while the 35 K protein present in the conditioned media of over-confluent cells may be a marker of morphological transformation.