A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.

Nuclear targeting determinants of the phage P1 cre DNA recombinase.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes. Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization signal. Region II exhibits a beta-sheet structure with which the bipartite motif may interact. However, neither region is by itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been shown to carry determinants that allow it to target the eukaryotic nucleus.

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.

Characterization of monoclonal thyroid-stimulating and thyrotropin binding-inhibiting autoantibodies from a Hashimoto's patient whose children had intrauterine and neonatal thyroid disease.

A multiplicity of TSH receptor autoantibodies (TSHRAbs) have been characterized after subcloning heterohybridomas produced from the lymphocytes of a patient who has Hashimoto's thyroiditis and had three children with intrauterine or neonatal hyperthyroidism. Twelve clones produced stimulating TSHRAbs that increased cAMP levels and iodide uptake in rat FRTL-5 thyroid cells and increased cAMP levels in Chinese hamster ovary (CHO) cells transfected with the human TSHR; like 95% of Graves' stimulating TSHRAbs, all 12 have their functional epitope on the N-terminus of the TSHR extracellular domain, requiring residues 90-165 for activity. All 12 bind to human thyroid membranes in the absence, but not the presence, of TSH, but are only weak inhibitors of TSH binding in assays measuring TSH binding-inhibiting Igs (TBIIs). In contrast, 8 different clones produced TSHRAbs that did not increase cAMP levels, but, instead, exhibited significant TBII activity. Four inhibited the ability of TSH or a stimulating TSHRAb to increase cAMP levels and had their functional epitope on the C-terminal portion of the TSHR external domain, residues 261-370, mimicking the properties of blocking TSHRAbs that cause hypothyroidism in patients with idiopathic myxedema. The 4 other TBIIs inhibited the ability of TSH, but not that of a stimulating TSHRAb, to increase cAMP levels, like TBIIs in Graves' patients. The functional epitope for 3 of these Graves'-like TBIIs was residues 90-165; the functional epitope for the fourth was residues 24-89. The fourth also increased arachidonic acid release and inositol phosphate levels in FRTL-5 thyroid cells and exhibited conversion activity, i.e. the ability to increase cAMP levels in the presence of an anti-human IgG. Thus, this TBII exhibited signal transduction activity, unlike the other 3 Graves'-like TBIIs. The patient, therefore, has stimulating TSHRAbs and 3 different types of TBIIs, each with different functional properties and different epitopes on the TSHR.

Cysteine proteinases are responsible for characteristic transketolase alterations in Alzheimer fibroblasts.

Cultured fibroblasts from patients affected by Alzheimer's disease (AD) exhibited peculiar alterations of the enzyme transketolase (TK). Abnormalities (dubbed alkaline bands, ab) consisted of enzyme forms having unusually high pl and were proposed as a marker of the disease in living patients. The mechanisms of TK-ab expression were investigated with the use of cysteine proteinase inhibitors and purified preparations of either rat liver or human cysteine proteinases. The cysteine proteinase inhibitors N-acetyl-leu-leu-norleucinal (ALLN), L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane (E-64), and egg white cystatin added to AD cells just prior to extraction abolished TK abnormalities. Moreover, 1 day incubation of AD cultures with either ALLN (10 micrograms/ml), NH4Cl (10 mM), or KCl (30 mM) prevented TK-ab generation, due, presumably, to an impairment of lysosomal functions. Isolated rat liver cysteine proteinases were able to degrade TK in normal extracts and reproduce the characteristic TK-ab of AD fibroblasts. Moreover, pure human cathepsin H was also shown to partially induce an Alzheimer-like TK pattern and cleave normal TK to a 35 kDa fragment as spontaneously occurring in AD fibroblasts. The explanation of mechanisms of TK-ab formation provided evidence for an underlying imbalance of proteolysis in AD fibroblasts due to a relative increase/derangement of the cysteine proteinases cathepsins which might be also involved in the reported abnormal processing of multiple cellular components.

Characteristic transketolase alterations in dermal fibroblasts of Alzheimer patients are modulated by culture conditions.

Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline pI, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO2 saturation in the atmosphere and with 3.7 g/liter NaHCO3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.

Differences in K+ permeability between cultured adult and neonatal rat islets of Langerhans in response to glucose, tolbutamide, diazoxide, and theophylline.

The effects of glucose, tolbutamide, and diazoxide on K+ permeability in neonatal and adult rat pancreatic islets, maintained in culture 1 week, were investigated by measuring the 86Rb outflow rate from prelabeled islets. In the absence of glucose, the 86Rb efflux was significantly lower in neonatal than adult islets. Raising the glucose concentration to 2.8, 5.6, 8.3, and 11.1 mM produced a marked reduction in the 86Rb efflux in adult islets but only a minor reduction in neonatal islets. The effect of tolbutamide to reduce, and diazoxide to increase, the 86Rb efflux was also less in neonatal islets. These results are discussed with respect to previously reported differences in insulin secretion from neonatal and adult islets in culture.

A goldfish model for evaluation of the neurotoxicity of omega-conotoxin GVI A and screening of monoclonal antibodies.

The neurotoxicity of omega-conotoxin (omega-CgTx), a potent neuronal voltage-sensitive calcium channel blocker, was measured using a new bioassay. omega-CgTx was administered intraperitoneally (ip) to goldfish weighing approximately 1.6 g, and dose-related changes were observed over a 2-hr period. omega-CgTx induced time- and dose-dependent abnormal swimming behavior (ASB) and mortality. The antitoxin activity of the antibodies was investigated in vivo by either (1) preincubation of the antibody with omega-CgTx at 4 degrees C overnight, or (2) pretreatment with antibody, 30 min before omega-CgTx injection in a 10:1 antibody/omega-CgTx molar ratio. The LD50 dose of omega-CgTx in goldfish was 5 nmol/kg ip, and preincubation of monoclonal antibody (50 nmol/kg ip) with omega-CgTx (5 nmol/kg ip) significantly (p less than 0.05) reduced mortality, ASB, and toxicity time. The antitoxin activity of the monoclonal antibodies evidenced in the goldfish bioassay was further tested in the conscious rat. In the rat, the increases in mean arterial pressure and heart rate induced by omega-CgTx (0.03 nmol/rat icv) were significantly (p less than 0.02 and p less than 0.01, respectively) attenuated by preincubation of the toxin with the antibody (0.3 nmol/rat). We conclude that the goldfish bioassay provides a simple, accurate, and inexpensive in vivo model for the study of the toxicity of omega-CgTx.

Monoclonal antibodies against the presynaptic calcium channel antagonist omega-conotoxin GVI A from cone snail poison.

Monoclonal antibodies have been prepared against omega-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic omega-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block omega-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block omega-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.

Lipid characteristics of RSV-transformed Balb/c 3T3 cell lines with different spontaneous metastatic potentials.

To determine whether a metastatic phenotype may be correlated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18:1) and a decrease of arachidonic (20:4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18:1 and decrease of 20:4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells. Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.

Effects of triiodothyronine on insulin release from cultured neonatal and adult rat islets.

Insulin secretion from neonatal and adult rat islets maintained in culture for 7-9 days in the absence or in the presence of 10 nM T3 was measured. In both neonatal and adult islets T3 treatment tends to inhibit insulin secretion only in the absence or in the presence of low glucose concentrations.

Effects of triiodothyronine on insulin release from cultured neonatal and adult rat islets

Insulin secretion from neonatal and adult rat islets maintained in culture for 7-9 days in the absence or in the presence of nMT3 was measured. In both neonatal and adult islets T3 treatment tends to inhibit insulin secretion only in the absence or in the presence of low glucose concentrations

Effects of glucose on insulin release and 86Rb permeability in cultured neonatal and adult rat islets.

Glucose-induced insulin release and modifications in 86Rb outflow were studied in cultured neonatal and adult rat islets. The dose-response curve for neonatal islets was steeper than for adult islets and the maximal response was clearly shifted towards lower glucose concentrations. In neonatal islets, glucose-induced insulin release was inhibited by the Ca2+-channel blocker, nifedipine. In the absence of glucose, the 86Rb outflow from neonatal islets was lower than from adult islets. Also, the glucose-induced reduction in 86Rb outflow was less pronounced in neonatal islets. Altered K+ permeability in the B-cell membrane could explain the change in glucose sensitivity of neonatal islets.

Lipid composition of cultured B16 melanoma cell variants with different lung-colonizing potential.

Lipid components influence several cell surface properties that are critical in different stages of the metastatic process. In this study, we examined whether the different lung-colonizing potential of B16-F1 and B16-F10 melanoma cells could be related to a characteristic lipid profile. The lipid analyses, carried out on the same cell cultures used for the assay of lung-colonizing potential, revealed characteristics in the lipid composition of both B16-F1 and B16-F10 melanoma cells that are common to other systems of malignant cells: a high level of 18:1 associated with low proportions of polyunsaturated fatty acids in phospholipids, accumulation of ether-linked lipids and absence of complex gangliosides. The two B16 melanoma variants differed significantly only with respect to ether-linked lipids, due to a higher level of alkyl-PC in B16-F10 than in B16-F1.

Interaction of the thyrotropin receptor on rat FRTL-5 thyroid cells with thyrotropin and a thyrotropin-stimulating autoantibody from Graves' patients.

FRTL-5 rat thyroid cells were either surface-labeled with 125I or biosynthetically labeled with [3H]N-acetylglucosamine, solubilized by lithium diiodosalicylate and immunoprecipitated after sequential exposure to bovine thyrotropin and anti-bovine thyrotropin. Autoradiography of polyacrylamide gels run under denaturing conditions and in the presence of a reducing agent revealed two prominent bands with approximate molecular weights of 66-70 kDa and 47 kDa. Immunoprecipitation of the same radiolabeled and solubilized membrane preparations with a Graves' disease IgG having thyroid stimulating but no thyrotropin-binding inhibiting activity revealed only one major band, migrating near the 47 kDa component reactive with thyrotropin. No bands were immunoprecipitated in control incubations using normal human IgG or substituting radiolabeled, solubilized membranes from a rat thyroid cell line with no thyrotropin receptor activity. Thin layer chromatography of Folch extracts of the [3H]-N-acetylglucosamine-labeled immunoprecipitates obtained by either procedure indicated that a specific thyroid ganglioside was coprecipitated with the immunoprecipitated proteins in both cases.

Iodine suppression of iodide uptake in FRTL-5 thyroid cells.

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.

Monoclonal antibody studies defining the origin and properties of autoantibodies in Graves' disease.

The present report summarizes experiments with monoclonal antibodies to the TSH receptor. The data provide further insight into the TSH receptor structure and into the basis of autoimmune antibodies implicated in the pathogenesis of Graves' disease. They resolve many clinical questions and provide new approaches to enhance our understanding of autoimmune disease. In one new approach, it has been noted that the 11E8 TBIAb can precipitate the phosphorylated beta subunit of the insulin and IGF1 receptor. This cross-reactivity or recognition of determinants adjacent to the TSH receptor may not be random. Insulin, IGF1, alpha 1 adrenergic, and TSH receptors have been linked to a synergistic cascade response system of the thyroid involving growth, thyroglobulin biosynthesis, iodination of thyroglobulin, and thyroid hormone formation. Future studies with the monoclonals may help unravel this cascade system and its regulatory relationships, along with the relationships between autoimmune thyroid disease and autoimmune diseases of other organs.

Ganglioside composition of substrate-adhesion sites of normal and virally-transformed Balb/c 3T3 cells.

The ganglioside composition of the so-called substrate-attached material (SAM), which remains tightly bound to the tissue culture dish after cells are detached by chelating agents, was compared with the ganglioside composition of released cell bodies in the cultures of normal and various virally-transformed Balb/c 3T3 cells. Regardless of whether the cells were untransformed or transformed, the SAM of their cultures shows a ganglioside structure characterized by a prevalence of the higher homologs, mainly GD1a, over the simpler gangliosides, even when the level of higher homologs was reduced in the cell bodies of transformed cells. This result cannot be ascribed to the presence of plasmamembranes in the SAM as shown by ganglioside analysis of the plasmamembranes of some of the cells under study. Only in a highly metastatic transformed cell line did the SAM contain the same low GD1a level as found in the cell bodies.