Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

Response of a human head/neck/upper-torso replica to dynamic loading--I. Physical model

A human head/neck/upper-torso replica was constructed and instrumented and its response to impact and dynamic loading was studied. The model consists of a water-filled cadaver skull; plastic vertebrae, sternum and ribs; silicon rubber disks and ligaments; and fabric muscles. The static behavior of the system under sagittal plane and lateral loading was adjusted so as to correspond to that of cadaver behavior under similar loading. The structure was loaded impulsively by the sudden arrest of a supporting sled running on a track and by direct head impact with a suspended steel ball. The measured response included the head acceleration, the disk pressures, the muscle strains, the intracranial pressures and the skull strains; the sled motion was also monitored. These data were recorded with a microcomputer and oscilloscopes; the overall system deformation was observed by high-speed cameras. The muscle contraction effects were determined with the aid of microcomputer-controlled devices including a vacuum system, solenoid valves and plastic syringes.     

Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membrane-associated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64-73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC-PK1 cultures after HMBA treatment is localized in cells forming a three-dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100-110 kD both in LLC-PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'-dimethylformamide (DMF) and equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound-repair of the monolayer. In addition to the membrane-bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers. These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.     

Induction of alkaline phosphatase in primary cultures of epiphyseal growth plate chondrocytes by a serum-derived factor

Alkaline phosphatase (AP) activity in epiphyseal growth plate cartilage increases markedly during differentiation of the chondrocytes, and reaches high levels in the zone of hypertrophy where vascular penetration and provisional mineralization begin. A proteinaceous factor has been discovered in serum that stimulates the expression of AP in chicken growth plate chondrocytes when these cells are grown in serum-free media. Sera from a variety of vertebrate species (goat, fetal bovine, horse, human, and chicken) all contained detectable levels of the inducing activity. The chondrocyte AP-induction factor (CAP-IF) from fetal bovine serum was precipitated with ammonium sulfate between 33% and 50% saturation, and purified by dye-ligand affinity chromatography. The active fraction, which eluted from an Affi-Gel Blue column between 0.10 and 0.15 M NaCl, was further resolved on a QMA anion exchange column. The most active and almost homogeneous fraction contained primarily a 64.5 kDa protein; about 3 micrograms/ml medium induced 50% of the maximal level of AP induction. CAP-IF is stable to heat (100 degrees C for 3 min) and dithiothreitol (50 mM) treatment, and is only mildly inactivated by 2 h treatment with trypsin. CAP-IF caused no significant effect on cell division as measured by 3H-thymidine uptake. Time-course studies revealed that at least 18-24 h exposure of the chondrocytes to CAP-IF is required to produce major increases in AP activity. Longer exposure time generally further increases the response. Cycloheximide almost completely blocked the increase in AP activity, indicating that de novo protein synthesis is required for induction.     

The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis

The aacA-aphD aminoglycoside resistance determinant of the Staphylococcus aureus transposon Tn4001, which specifies resistance to gentamicin, tobramycin and kanamycin, has been cloned and shown to express these resistances in Escherichia coli. The determinant encoded a single protein with an apparent size of 59 kDa which specified both aminoglycoside acetyltransferase [AAC(6')] and aminoglycoside phosphotransferase [APH(2")] activities. Nucleotide sequence analysis of the determinant showed it to be capable of encoding a 479-amino-acid protein of 56.9 kDa. analysis of Tn1725 insertion mutants of the determinant indicated that resistance to tobramycin and kanamycin is due to the AAC activity specified by, approximately, the first 170 amino acids of the predicted protein sequence and is consistent with the gentamicin resistance, specified by the APH activity, being encoded within the C-terminal region of the protein. Comparison of the C-terminal end of the predicted amino acid sequence with the reported sequences of 13 APHs and a viomycin phosphotransferase revealed a region which is highly conserved among these phosphotransferases.     

Investigation of variation in phenotype and DNA content between single-conidium isolates of single Penicillium strains

Variation in phenotypic properties was examined in three strains of closely related fasciculate species of Penicillium using 114 morphological, physiological, and biochemical characters. Thirty-six of these characters showed variation within single-conidium isolates of the same strain. Conidial sizes and nuclear DNA contents were compared using flow microfluorimetry; these results suggested that significant differences in conidial DNA content are associated with phenotypic variation. The taxonomic significance of the results is discussed.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.