Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.      

Synthetic signal peptide and analogs display different activities in mammalian and plant in vitro secretion systems

The biological properties of four chemically synthesized signal peptides were compared in mammalian (rabbit reticulocyte) and plant (wheat germ) cell-free protein secretion systems. The precursor-specific region of bovine pre-proparathyroid hormone (preproPTH), [D-Tyr-(+1)]preproPTH-(-29-+1)amide, and a sulfur-free analog, [Nle-(-25), Nle-(-21), Nle-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibit the processing of an unrelated precursor protein (pre-prolactin) to its mature secreted form (prolactin) in the mammalian system. In the plant system supplemented with signal recognition particle, the signal peptides arrest translation of both secretory (preprolactin) and cytoplasmic (globin) proteins. One analog, [Nle-(-25), Nle-(-21), Asp-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibits preprotein processing in the mammalian system but fails to induce translation arrest in the plant system. A truncated peptide, [N alpha-AcLeu-(-17), Ala-(-14), D-Tyr-(+1)]preproPTH-(-17-+1)amide, lacking the N-terminal (positively charged) region and a portion of the hydrophobic core region, is inactive in both systems. These studies demonstrate that the chemically synthesized signal region of a precursor protein interacts directly with signal recognition particle and functionally mimics the proposed properties of a native signal sequence linked to a nascent protein as it emerges from the ribosome during biosynthesis, and an analog of the signal peptide reveals fundamental differences between the components involved in the protein secretion apparatus in mammals and plants.     

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.     

Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.     

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.     

Morphology of connective tissue in skeletal muscle

The arrangement and distribution of connective tissue in six different skeletal muscles and smooth muscle was examined by scanning electron microscopy. The endomysial arrangement of collagen was similar in all types of muscle and consisted of three components: (1) myocyte-myocyte connectives; (2) myocyte-capillary connectives; and (3) a weave network of collagen intimately associated with the basal laminae of the myocytes. The perimysium of the different muscles was qualitatively similar but quantitatively dissimilar. The perimysium consisted of large tendon-like bundles of interwoven collagen which connected with the dense weave collagen that surrounded groups of muscles. The arrangement of the collagen in the perimysium and endomysium would explain differences in the mechanical properties of the different muscle. The contribution of the connective tissue to mechanical properties of muscle is discussed.     

STUDIES ON PROTEIN UPTAKE BY ISOLATED TUMOR CELLS : I. Electron Microscopic Evidence of Ferritin Uptake by Ehrlich Ascites Tumor Cells

Ferritin, added to the incubation medium of ascites tumor cells, was used as an electron microscopic marker to study the uptake of large protein molecules by morphologically intact cells. A definite uptake could be detected after 1 hour of incubation in Tyrode bicarbonate solution containing 0.04 to 13.3 mg ferritin/ml. Ferritin was found in a variety of membrane-surrounded structures, suggesting that pinocytesis and related membrane movements are occurring under physiological conditions and can account for the penetration of intact macromolecules into isolated tumor cells. Supplementation of the medium with serum albumin (33 mg/ml) increased the average amount of ferritin per cell and per pinocytotic structure. Ferritin was strongly adsorbed by fragments of lysed cells, which were readily taken up by intact cells. Besides its role as carrier, this debris appeared to stimulate membrane movements. Only rare examples were found to suggest the release of ferritin from the pinocytotic structures into the cytoplasm. Thus, the disintegration of such structures cannot be considered an obvious step towards a rapid metabolic utilization of protein by the cell. Particles of colloidal gold presented to the cell under the same conditions were not taken up to any significant extent, thus providing good evidence for a selective ingestion of particles of comparable sizes.     

Ultrastructural localization of giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and deduced protein sequence of alpha giardin

The giardins are a group of 29-38-kD proteins in the ventral disk of the protozoan parasite Giardia lamblia. The disk attaches the parasite to the host's intestinal epithelium and is composed of parallel, coiled microtubules that are adjacent to the ventral plasma membrane and from which processes called microribbons extend into the cytoplasm; the microribbons are connected by crossbridges. G. lamblia cytoskeletons, consisting of disks and attached flagella, were isolated and used to show that the 29-38-kD proteins separate into five bands by one-dimensional electrophoresis and into 23 species by two-dimensional analysis. Rabbit antibodies raised against a 33-kD protein band, purified by one-dimensional gel electrophoresis and shown to contain three proteins by two-dimensional electrophoresis, recognized 17 proteins by two-dimensional immunoblot analysis. By immunofluorescence these antibodies reacted with the ventral disk but not with the flagella in isolated cytoskeletons. Electron microscopy revealed that the anti-giardin antibodies bound to the edges of the microribbons but not to the microtubules, crossbridges, or other, nondisk structures. Antibodies to tubulin reacted with both the disk and flagella in isolated cytoskeletons but bound only to the microtubules in these structures. The amino-terminal sequence of the 33-kD immunogen was determined and used to construct a DNA oligomer, and the oligomer was used to isolate the alpha giardin gene. The gene was used to hybrid select RNA, and the in vitro translation product from this RNA was precipitated by the antibodies against the 33-kD immunogen. The gene sequence was a single open reading frame of 885 nucleotides that predicted a protein of 33.8 kD. The protein sequence is unique, having no significant homology to two other giardin sequences or to any sequences within the Protein Identification Resource. It is predicted to be 82% alpha helical. The downstream sequence of the gene indicates that the sequence AGT-PuAA is located six to nine nucleotides beyond the stop codon in all protein-encoding genes of G. lamblia that have been sequenced and reported to date.     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.