Usefulness of muscle denervation as an MRI sign of peripheral nerve pathology.

Peripheral nerve disorders may be classified into compressive or entrapment neuropathies and non-compressive neuropathies. Muscle denervation recognized on MRI may be a useful sign in the diagnosis of peripheral nerve disorders. Acute or subacute denervation results in prolonged T2 relaxation time, producing increased signal in skeletal muscle on short tau inversion-recovery and fat-suppressed T2-weighted images. Chronic denervation produces fatty atrophy of skeletal muscles, resulting in increased muscle signal on T1-weighted images. This review will outline and illustrate the various ways that muscle denervation as seen on MRI may assist in the diagnosis and localization of peripheral nerve disorders.

Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol).

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.

Surface-associated serum proteins inhibit the uptake of phosphatidylserine and poly(ethylene glycol) liposomes by mouse macrophages.

Serum proteins, acting as opsonins, are believed to contribute significantly to liposome-macrophage cell association and thus regulate liposome uptake by cells of the mononuclear phagocytic system (MPS). We studied the effect of serum protein on binding and uptake of phosphatidylglycerol-, phosphatidylserine-, cardiolipin-, and N,N-dioleyl-N,N-dimethylammonium chloride- (DODAC) containing as well as poly(ethylene glycol)- (PEG) containing liposomes by mouse bone marrow macrophages in vitro. Consistent with the postulated surface-shielding properties of PEG, protein-free uptake of liposomes containing 5 mol% PEG and either 20 mol% anionic phosphatidylserine or 20 mol% cationic DODAC was equivalent to uptake of neutral liposomes. In contrast to previous reports indicating that protein adsorption to liposomes increases uptake by macrophages, the presence of bound serum protein did not increase the uptake of these liposomes by cultured macrophages. Rather, we found that pre-incubating liposomes with serum reduced the uptake of liposomes containing phosphatidylserine. Surprisingly, serum treatment of PEG-containing liposomes also significantly reduced liposome uptake by macrophages. It is postulated that, in the case of phosphatidylserine liposomes, the bound serum protein can provide a non-specific surface-shielding property that reduces the charge-mediated interactions between liposomes and bone marrow macrophage cells. In addition, incubation of PEG-bearing liposomes with serum can result in a change in the properties of the PEG, resulting in a surface that is better protected against interactions with cells.

In vitro characterization of the anticancer activity of membrane-active cationic peptides. I. Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines.

Cationic amphipathic peptides, such as the defensins and cecropins, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability. Increased permeability may lead to cell lysis or, alternatively, may produce subtle changes in the membrane's barrier function that promote cell death. The in vitro cytotoxic and lytic activity of short mammalian-derived extended-helical cationic peptides and insect-derived alpha-helical peptides was measured in this study with the objective of establishing the anticancer potential of these agents. Two specific aims were addressed: (i) to assess the activity of peptides against non-malignant cells (sheep erythrocytes and human umbilical vein endothelial cells) versus tumor cells; and (ii) to characterize the cytotoxic activity using multidrug-resistant tumor cell lines in the presence and absence of the anthracycline doxorubicin. Cell lysis assays demonstrated that the lytic activity of the peptides tested was 2->50 times more cytotoxic to tumor cells than to non-malignant cells. Further, the cytotoxic activity of these peptides was equivalent when tested against sensitive and multidrug-resistant cell lines. In addition to their inherent cytotoxic activity, these membrane-active peptides can also augment the in vitro cytotoxic activity of doxorubicin against multidrug-resistant tumor cells.

Regulation of annexin I-dependent aggregation of phospholipid vesicles by protein kinase C.

Annexin I is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins. The ability of this protein to aggregate and to mediate the fusion of various types of vesicles has supported the hypothesis that this protein might be involved in intracellular membrane fusion processes such as exocytosis. Although annexin I has been described as a major in vitro substrate of both protein kinase C and the epidermal-growth-factor-receptor protein tyrosine kinase, the functional consequences of these phosphorylation events have not been investigated. In this paper we examine the effect of the phosphorylation of annexin I by protein kinase C on the phospholipid aggregation activity of the protein. The stoichiometry of phosphorylation of the protein was affected by the method of preparation of the phospholipid. Under optimal assay conditions protein kinase C catalysed the incorporation of 2.83 +/- 0.23 mol of phosphate/mol of annexin I (mean +/- S.E.M., n = 21). Studies with the Ca(2+)- and phospholipid-independent form of protein kinase C suggested that the phosphorylation of annexin I was stimulated by phospholipid in the absence of Ca2+, although maximal phosphorylation was achieved in the presence of both phospholipid and Ca2+. Phosphorylation of annexin I resulted in a dramatic decrease in the rate and extent of phospholipid vesicle aggregation, without significantly disrupting the binding of the protein to the phospholipid vesicles. The phosphorylation of annexin I increased the EC50 (Ca2+) of phospholipid vesicle aggregation from 19 +/- 10 microM (mean +/- S.D., n = 7) for the native protein to 290 +/- 95 microM (mean +/- S.D., n = 5) for the phosphorylated protein. These results suggest that protein kinase C may act to inhibit the phospholipid vesicle aggregation activity of annexin I.

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.

Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.

Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase.

Autoantibodies to cellular Ag are found in the sera of patients with systemic rheumatic diseases. Identification and characterization of the reactive autoantigens has helped clinicians to define subsets of rheumatic diseases and has assisted biologists in defining the function within the cell of these molecules. We have studied autoantibodies from patients that react with the centrosome (centriole) region of the cell. We found by immunoblotting techniques that these antibodies react with a 48-kDa protein. Additional immunoblotting and affinity purification studies indicate that the Ag may be the glycolytic enzyme enolase.

Membrane glycoprotein and surface free energy changes in hypoxic fibroblast cells.

Hypoxia affects the biochemistry of mammalian cells and thus alters their sensitivity to subsequent chemo- and radiotherapy. When V79 Chinese hamster lung fibroblasts were grown under conditions of extreme hypoxia (less than 10 ppm O2) there was a significant shift in the membrane glycoprotein composition. Scanning electron microscopy revealed altered cell surface morphology including loss of pseudopodial projections. Experiments to determine changes in interfacial free energy of these cells using equilibrium two phase systems of poly(ethylene glycol) (PEG) and dextran were carried out. Test fluid droplets of the denser dextran-rich phase were formed on layers of cells in the PEG-rich phase as the bathing medium, and the contact angles the droplets made with the cell layers were measured from photomicrographs. The contact angles on cells in the plateau phase increased significantly with time of exposure to hypoxia, from 25 degrees (zero time) to 35 degrees (6 h) to 60 degrees (9 h). Contact angles on cells in the exponential phase increased from 80 degrees (zero time) to 150 degrees after 20 h of hypoxia. It appears that the altered contact angles reflect changes in cell surface hydrophobicity that may, in part, reflect alterations in the membrane glycoprotein composition.