Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.     

Modulation of EGF binding and action by succinylated concanavalin A in fibroblast cell cultures

The role of the binding of succinylated concanavalin A to tissue culture cells in influencing epidermal growth factor (EGF)-mediated cell proliferation has been studied. Succinylated concanavalin A dramatically reduces the stimulation of 3T6 cells by EGF in Dulbecco's modified Eagle's medium (DME) containing insulin and vitamin B12 as additional growth factors, but no serum. Furthermore, binding studies using 125I-labeled EGF have shown that the binding of EGF to the cell surface is reduced upon addition of succinylated concanavalin A.     

Direct linkage of EGF to its receptor: characterization and biological relevance

A small portion of the 125I-EGF that binds specifically to intact cells or isolated membrane from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which possesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for information of each is similar, showing that 125I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to 125I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of 3H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.     

Identification of the estrogen receptor Cd-binding sites by chemical modification

The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium. In the present study we utilized the rainbow trout ER ligand-binding domain fused to glutathione-S-transferase, and used Cd-shielding against various types of chemical modification of the fusion protein to study non-covalent interactions between the ER and Cd. The distribution of exposed and shielded residues allowed to identify amino acid residues involved in the interaction. Our data indicated preferential protection of Cys groups by cadmium, suggesting their involvement in the interaction. This supports data found in the literature on the strong binding affinity of the thiol group towards metals. However, not all Cys in the fusion protein sequence were protected against chemical modification, illustrating the importance of their chemical environment. In general, the location of rtER-LBD Cys residues implicated in Cd interactions did not confirm assignments made by alanine-scanning mutagenesis for the hER, probably due to differences in experimental setup and fusion proteins used. The involvement of other functional groups such as carboxylic acids in the Cd interactions, though not confirmed, can not be completely ruled out due to the general limitations of the chemical modification approach discussed in detail. Suggestions for an improved experimental setup were made.     

Synergistic action of phenolic signal compounds and carbohydrates in the induction of virulence gene expression of Agrobacterium tumefaciens.

Virulence (vir) gene expression of Agrobacterium tumefaciens is activated by plant phenolic compounds such as alpha-hydroxyacetosyringone (HOAS), acetosyringone (AS), methyl syringate, coniferyl alcohol and sinapyl alcohol. Inositol was found to be a potentiating factor of vir-inducing activity, which enhanced the vir-inducing activity of AS and HOAS in a synergistic manner, in particular at a low concentrations of AS and HOAS. Of the other sugars tested D-glucose, L-rhamnose, D-xylose and D-galacturonic acid, the main components of plant cell wall polysaccharides, remarkedly potentiated the vir-inducing activity of AS, indicating the cooperative action of the signal compounds and sugars in Agrobacterium infection to plants.     

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.     

Liquid-crystalline phases of circular superhelical plasmid DNA and their modification by the action of nuclease enzymes

CD spectra of liquid-crystalline dispersions, X-ray diffraction patterns and optical textures of liquid crystals prepared from native superhelical DNA in poly(ethyleneglycol)-containing water-salt solutions before and after treatment of DNA with micrococcal nuclease have been obtained. It was found that condensation of native superhelical DNA is accompanied by the formation of liquid crystals with a non-specific optical texture. After treatment of the DNA, liquid-crystalline dispersions, with Micrococcal nuclease the DNA is able to form two similar types of liquid crystals with abnormal optical activity which differ in the peculiarities of their textures. The data obtained demonstrate the formation of multiple types of liquid crystals from high molar mass double-stranded optically active DNA molecules.     

Proteasomes: the changing face of proteolysis

The proteasome, the multimeric, multicatalytic complex that is responsible for much of the proteolysis within cells, catalyzes several different types of peptide cleavage. Recently, it has been found to increase its repertoire of catalytic activities even further, by varying its catalytic subunits and by associating with regulatory complexes.     

The antiulcer action of sophora and the active constituent in Sophora. II. The antiulcer action of vexibinol

The inhibitory effect of vexibinol, one of the flavanols found in Sophora, on gastric ulcers induced by HCl-ethanol has been reported previously. In the present study, the effect of vexibinol was examined in various experimental ulcer models in order to determine the mechanism of its antiulcer action. The results indicated that the oral administration of vexibinol at 25-50 mg/kg significantly inhibited the development of ulcers induced by HCl-ethanol, 0.6 N HCl 0.2 N NaOH, absolute ethanol and 1% NH3. In addition, an intraduodenal administration of vexibinol at 300 mg/kg significantly inhibited Shay's ulcer. Further, intraduodenal administration at 300 mg/kg significantly inhibited acid secretion caused by 2-deoxy-D-glucose. These results suggest that vexibinol has not only gastric mucosal protective action but also an inhibitory effect on the secretion of gastric acids.     

The amyloidogenicity of gelsolin is controlled by proteolysis and pH.

BACKGROUND: Normally, gelsolin functions in plasma as part of the actin-scavenging system to assemble and disassemble actin filaments. The Asp 187-->Asn (D187N) Asp 187-->Tyr (D187Y) gelsolin mutations facilitate two proteolytic cuts in the parent protein generating a 71-residue fragment that forms amyloid fibrils in humans, putatively causing Finnish type familial amyloidosis (FAF). We investigated the role of the D187N mutation in amyloidogenicity using biophysical studies in vitro. RESULTS: Both the recombinant wild-type and D187N FAF-associated gelsolin fragments adopt an ensemble of largely unfolded structures that do not self-associate into amyloid at pH 7. 5. Incubation of either fragment at low pHs (6.0-4.0) leads to the formation of well-defined fibrils within 72 hours, however. CONCLUSIONS: The D187N mutation has been suggested to destabilize the structure of the gelsolin parent protein (specifically domain 2), facilitating two proteolytic cleavage events. Our studies demonstrate that generating the largely unstructured peptide is not sufficient alone for amyloid formation in vitro (on a time scale of months). A drop in pH or an analogous environmental change appears necessary to convert the unstructured fragment into amyloid fibrils, probably through an associative mechanism. The wild-type gelsolin fragment will make amyloid fibrils from pH 6 to 4 in vitro, but neither the wild-type fragment nor fibrils have been observed in vivo. It is possible that domain 2 of wild-type gelsolin is stable in the context of the whole protein and not susceptible to the proteolytic degradation that affords the 71-residue FAF-associated peptide.     

Chemical modification of polymers. XII. Control of triboelectric charging properties of polymers by chemical modification

Chemical modification of polymers leads to changes in triboelectric charging properties which are proportional to the degree of conversion. This contrasts to physical mixtures in which the surface energy causes one component to dominate the surface, hence the charging properties. Relationships between molecular structure and triboelectric charging have been deduced from the results. Thus the direction of change of triboelectric charging on chemical modification is governed by the nature of the reaction and the magnitude of the change is governed by the extent of reaction.     

Chemical modification of polynorbornene. I. Sulfonation in dilute solution

The sulfonation of dilute solutions of polynorbornene (PN) in CCl₄ has been carried out using SO₃–triethylphosphate complexes of various stoichiometries dissolved in dichloroethane (DCE) or trifluoro-trichloro-ethane (TTE). This leads to an electrophilic substitution, with very few side reactions, yielding high sulfonation ratios (ca. 85%). The initial reaction is first order in both reactants (PN and SO₃) and has an activation energy of 17.5 kJ mol^?1. The reaction becomes diffusion-controlled after ca. 30 min at room temperature due to the precipitation of the partially sulfonated polymer. The initial rate is almost independent of the isomeric structure (cis or trans) and the molecular weight of the polymer, as well as on the stoichiometry of the SO₃–TEP complex; it is higher when TTE is used as solvent. The final sulfonation ratio is strongly dependent on the concentration of SO₃ and seems to be controlled by the conditions of diffusion inside the precipitated solid.     

Phase transfer catalysis in the chemical modification of polymers. Part II

A soluble partly chloromethylated polystyrene has been successfully modified by compounds containing labile hydrogen and by NaCN or NaSCN, under PTC conditions.     

Molecular dynamics characterization of the structures and induction mechanisms of a reverse phenotype of the tetracycline receptor

Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.     

Damping and modification of the multiquanta Davydov-like solitons in molecular chains.

Relaxation of the multivibron soliton in molecular chain on lattice vibrations is investigated within the simple microscopic model. It was shown that its dynamics is governed by the nonlinear Schr?dinger equation containing damping term. Its appearance is the consequence of the emission and absorption of real phonons arising when soliton velocity approaches the phase speed of sound. Explicit time dependence of the soliton parameters results as a consequence of these perturbations. In particular, soliton amplitude decreases while its width increases.     

Light-induced proteolysis of myosin heavy chain by Rose Bengal-conjugated antibody complexes.

The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS-PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.