Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

A quantitative model for partition in aqueous multiphase systems.

A model for the partition of charged molecules in aqueous multiphase systems has been developed. The partition coefficient of one component, or the overall partition coefficient of a number of components, between two arbitrary phases is expressed in terms of the difference in electrical potential between the phases (due to electrolytes present in the system), the net charges of the partitioned components and their partition coefficients in a (sometimes hypothetical) uncharged state. The fraction of material in one phase has also been described as a function of the net charges of the partitioned components. The model fits well to experimental data for partition of chromate, pyridine, ribonuclease A, two types of CO-hemoglobin and an enzyme mixture (yeast lysate) in three-phase systems consisting of poly(ethylene glycol), dextran, Ficoll and water. Minor deviations from the model are construed to be a pH-dependent uptake of ions. The data have also been used to detect differences in solvation of similar proteins, as well as the presence of several forms of some glycolytic enzymes present in yeast lysate.     

Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?

The gene for the chromosomally encoded dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990 has been cloned and characterized. The structural gene encodes a polypeptide of 161 amino acid residues with a calculated molecular weight of 18,417. This trimethoprim-sensitive (Tmps) DHFR, SeDHFR, differs in only three amino acids (Val-31-->Ile, Gly-43-->Ala, and Phe-98-->Tyr) from the trimethoprim-resistant (Tmpr) S1 DHFR encoded by transposon Tn4003. Since in addition the S. epidermidis gene also forms part of an operon with thyE and open reading frame 140 as in Tn4003, the chromosomally located gene encoding the Tmps SeDHFR is likely to be the molecular origin of the plasmid-located gene encoding the Tmpr S1 DHFR. Site-directed mutagenesis and kinetic analysis of the purified enzymes suggest that a single Phe-->Tyr change at position 98 is the major determinant of trimethoprim resistance.     

Infection of the heart cockle, Clinocardium nuttallii, from Yaquina Bay,Oregon, with an endosymbiotic alga

Clinocardium nuttallii from Yaquina Bay, Oregon, were found to harbor an endosymbiotic alga. Some aspects of this relationship are presented. The areas of infection include the siphon, mantle, and occasionally the foot. C. nuttallii under 2 years old are not infected; the incidence and intensity of the infection increases in the older age groups. For all ages the incidence averages 35%. Pigment composition, morphology, and growth characteristics of the alga are similar to those found in the genus Chlorella. In situ, the algae form dense, sometimes massive colonies but do not appear to cause any host reaction or enfeeblement. The thick layers surrounding the algal cells in situ, the dense colonies, and the in vitro reaction to host extract suggest the alga is parasitic. However, the presence of chloroplasts, the location of the algal cells only in illuminated tissue, the seemingly unhampered reproduction in situ, and the eventual adaptation to mineral medium suggest the alga is a facultative parasite. Experimental infection was achieved by feeding mature uninfected cockles a diet of only symbiont cells. In vitro observations found the symbiont cells readily engulfed by host blood amoebocytes. It is believed that the animal acquires the infection through phagocytosis of the symbiont cells and subsequent diapedesis across epithelial barriers by host amoebocytic cells.     

Does high-mobility-group non-histone protein HMG 1 interact specifically with histone H1 subfractions?

The interaction of the non-histone chromosomal protein HMG (high-mobility group) 1 with histone H1 subfractions was investigated by equilibrium sedimentation and n.m.r. sectroscopy. In contrast with a previous report [Smerdon & Isenberg (1976) Biochemistry 15, 4242--4247], it was found, by using equilibrium-sedimentation analysis, that protein HMG 1 binds to all three histone H1 subfractions CTL1, CTL2, and CTL3, arguing against there being a specific interaction between protein HMG 1 and only two of the subfractions, CTL1 and CTL2. Raising the ionic strength of the solutions prevents binding of protein HMG 1 to total histone H1 and the three subfractions, suggesting that the binding in vitro is simply a non-specific ionic interaction between acidic regions of the non-histone protein and the basic regions of the histone. Protein HMG 1 binds to histone H5 also, supporting this view. The above conclusions are supported by n.m.r. studies of protein HMG 1/histone H1 subfraction mixtures. When the two proteins were mixed, there was little perturbation of the n.m.r. spectra and there was no evidence for specific interaction of protein HMG 1 with any of the subfractions. It therefore remains an open question as to whether protein HMG 1 and histone H1 are complexed together in chromatin.     

Non-peptide alpha(v)beta(3) antagonists. Part 3: identification of potent RGD mimetics incorporating novel beta-amino acids as aspartic acid replacements.

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.     

Donor-site morbidity after free vascularized autogenous fibular transfer: subjective and quantitative analyses

The purpose of this study was to determine the subjective and quantitative donor-site morbidity after removal of a free vascularized fibula flap for autoreconstruction. Ten patients and six age-matched, healthy control subjects were included in this study. The postoperative periods ranged from 6 to 87 months. Subjective donor-site morbidity was assessed with a patient questionnaire and the Enneking system. For quantification of donor-site morbidity, gait was evaluated during normal walking, walking under visual and cognitive constraints, and walking at a velocity higher than the preferred one. In general, the patient perception of donor-site morbidity was low. Complaints were frequently mentioned, however, including pain (60 percent), dysesthesia (50 percent), a feeling of ankle instability (30 percent), and inability to run (20 percent). Gait analyses revealed that patients walked at a lower preferred velocity, compared with control subjects. Furthermore, they demonstrated significant increases in the coefficients of variation of stride time during walking under visual and cognitive loads and during walking at a velocity higher than the preferred one, compared with normal walking. These increases were not observed for control subjects. These findings suggest that the reautomatization of gait is affected among patients. This study demonstrates that fibula harvesting is associated with low subjective morbidity but frequent complaints. Walking during complex tasks and at high velocities reveals that restoration of gait is not complete after partial fibulectomy.