Effects of ultra-wideband electromagnetic pulses on pre-neoplastic mammary epithelial cell proliferation.

Electromagnetic ultra-wideband pulses (UWB) or nanopulses, are generated by a wide range of electronic devices used in communications and radar technology. However, the specific effects of nanopulse exposure on cell growth and function have not been extensively investigated. Here, studies have been conducted to determine the effects of prolonged exposure to non-ionizing, low to moderate intensity nanopulses on the growth of pre-neoplastic CL-S1 mammary epithelial cells in vitro. Cells were grown in culture and maintained in serum-free defined medium containing 10 ng/ml EGF and 10 microg/ml insulin as comitogens. Studies showed that 0.25-3.0 h exposure to nanopulses of 18 kV/m field intensity, 1 kHz repetition rate and 10 ns pulse width had no effect on CL-S1 cell growth or viability during the subsequent 72-h culture period. However, exposure to similar nanopulses for prolonged periods of time (4-6 h) resulted in a significant increase in cell proliferation, as compared to untreated controls. Additional studies showed that nanopulse exposure enhanced CL-S1 cell growth when cells were maintained in media containing only EGF, but had no effect on cells maintained in defined media that were mitogen-free or containing only insulin. Studies also showed that the growth-promoting effects of nanopulse exposure were associated with a relatively large increase in intracellular levels of phospho-MEK1 (active) and phospho-ERK1/2 (active) in these cells. These findings demonstrate that prolonged exposure to moderate levels of UWB enhanced EGF-dependent mitogenesis, and that this growth-promoting effect appears to be mediated by enhanced activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pre-neoplastic CL-S1 mammary epithelial cells.

Induced mitogenic activity in AML-12 mouse hepatocytes exposed to low-dose ultra-wideband electromagnetic radiation.

Ultra-wideband (UWB) technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR) could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM) cell. Cells were exposed to UWBR for 2 h at a temperature of 23 degrees C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05) dose-dependent response in cell viability in both serum-treated and serum free medium (SFM) -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

Determinants of the fidelity of processing glucoamylase-lysozyme fusions by Aspergillus niger.

Fusion proteins are used to enhance the yields of heterologous proteins secreted from filamentous fungi. In Aspergillus niger, the target protein is normally fused downstream of the carrier protein glucoamylase with a Lys-Arg KEX2-like cleavage site at the junction. This is cleaved in vivo to release mature protein but the processing is not always accurate. We have used N-terminal mutant lysozymes to vary the sequence immediately downstream of the KEX site, and also varied the amino acid sequence upstream of the KEX processing site, to study the fidelity of processing. The sequences both upstream and downstream of the KEX2 site affected the fidelity of cleavage. With some constructs, a range of processing sites were apparent and the relative proportions were time dependent in batch cultures of A. niger. Aberrant processing was related to the secondary-structure preferences of the amino acids in and around the KEX site. Downstream of the processing site, the fidelity of processing decreased in proportion to the tendency for helix formation.

Structural characterisation and comparison of the native and A-states of equine lysozyme.

Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.

The N-terminal domains of tensin and auxilin are phosphatase homologues.

Tensin, an actin filament capping protein, and auxilin, a component of receptor-mediated endocytosis, are known to have 350 residue regions of significant sequence similarity near their N-termini (Schröder et al., 1995, Eur J Biochem 228:297-304). Here we demonstrate that these regions are homologous, not only to each other, but also to the catalytic domain of a putative protein tyrosine phosphatase (PTP) from Saccharomyces cerevisiae and to other PTPs. We propose that the PTP-like portion of the homology region of tensin and auxilin represents a distinct domain. A detailed sequence comparison indicates that the PTP-like domain in tensin is unlikely to exhibit phosphatase activity, whereas in auxilin it may possess a different phosphatase specificity from tyrosine phosphatases. It is probable that the PTP-like domains in tensin and auxilin mediate binding interactions with phosphorylated polypeptides; they may therefore represent members of a distinct class of phosphopeptide recognition domain.

Structural basis of the stability of a lysozyme molten globule.

Hydrogen exchange measurements on equine lysozyme show that amides in three of the four major helices of the native protein are significantly protected in a molten globule state formed at pH 2. The pattern of protection within the different helices, however, varies significantly. Examination of the pattern in the light of the native structure indicates that the side chains of the protected residues form a compact cluster within the core of the protein. We suggest that such a core is present in the molten globule state, indicating the existence of substantial native-like interactions between hydrophobic residues. The formation of clusters of this type during the early stages of folding could be crucial to directing polypeptide chains to their native structures.

Conformational properties of four peptides spanning the sequence of hen lysozyme.

Four peptides encompassing the entire amino acid sequence of hen lysozyme were examined in aqueous solution and in 50% (v/v) 2,2,2-trifluoroethanol (TFE) by far-UV CD. Two peptides, 1-40 and 84-129, correspond to regions which are helical in the native protein, and together represent the alpha-domain. The beta-domain of the native enzyme was also synthesized as two peptides, one (41-60) containing the residues in the triple stranded antiparallel beta-sheet and the other (61-82) corresponding to a region lacking regular secondary structure. In water at pH 2.0 and 25 degrees C, the monomeric peptides 1-40, 41-60 and 61-82 appear to be predominantly unstructured. By contrast, the peptide 84-129 has considerable, presumably helical structure, corresponding to approximately 19%, or nine residues, on average, which can be unfolded by the addition of 8 M urea or 6 M guanidine hydrochloride. In 50% TFE the conformational properties of the four peptides are again distinct. Although little helical structure is induced in the peptides 41-60 and 61-82, and a native-like extent of helical structure is induced in the peptide 1-40, the peptide 84-29 converts almost entirely to helical structure in 50% TFE. The far-UV CD spectrum of a stoichiometric mixture of the four peptides in water resembles closely that of a denatured state of the intact protein formed by reductive methylation of its four disulphide bonds, but differs significantly from that of the native protein. The far-UV CD spectrum of the peptide mixture in TFE is indistinguishable from that of the intact protein in this solvent, both in the presence and in the absence of its four disulphide bonds. The conformational preferences of the peptides are not predicted using standard assessments of helical propensity or hydrophobicity, but correlate instead with the number of local contacts made in the native protein. On the basis of these results, we suggest that the region 84-129 could play an important role in determining the nature of the early folding events in the folding pathway of the intact polypeptide chain.

Thermodynamic strategies for stabilizing intermediate states of proteins.

This paper presents three theorems pertaining to thermodynamic properties of the intermediate (e.g., molten globule) state of proteins exhibiting such a conformation in the presence of GuHCl or urea. The theorems are proved for the three-state case using the denaturant binding model and the linear extrapolation model; their utility is illustrated via applications to examples in the literature. Theorem One states that the denaturant activity that maximizes the population of a partly folded conformation is at any temperature independent of the Gibbs free energy difference between the intermediate and native states. This result holds for both models of protein-denaturant interaction. The second theorem claims that the population maximum is independent of the denaturant association constant for the denaturant binding model. Theorem Three, which also applies to both models considered here, states that at the temperatures corresponding to the extrema in the population of the intermediate, the enthalpy change of the intermediate is equal to the excess enthalpy function, an experimentally accessible quantity. In the absence of denaturant, the enthalpy change of the intermediate state at the population extrema can be written as a function of the thermodynamic parameters of the unfolded state alone. These results, which can be applied to systems of any number of states under certain conditions, should aid in the optimization of conditions employed for experimental studies of partly organized states of proteins.

Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry.

We demonstrate a method for obtaining accurate estimates of the thermodynamic parameter values characteristic of a two-state folding/unfolding transition under conditions in which the onset of cold denaturation prevents the native state from being fully populated at any temperature. This situation occurs for proteins exhibiting low thermal stability, which may be intrinsic, the result of amino acid substitution, or the consequence of protein-solvent interactions (e.g., extremes of pH, the presence of denaturants, extremes of ionic strength). Conventional analysis of calorimetric scans obtained under such conditions yields erroneous values for the enthalpy, entropy, and heat capacity changes characteristic of the folding/unfolding transition of the protein. This paper describes for the first time the weighted average enthalpy function. In contrast to the van't Hoff and calorimetric enthalpies, the weighted average enthalpy yields thermodynamic parameter values which differ by less than 5% form the true value, even for situations in which the population of molecules in the native state at the start of the transition is half of the total.

Molecular basis of cooperativity in protein folding. IV. CORE: a general cooperative folding model.

The cooperative nature of the protein folding process is independent of the characteristic fold and the specific secondary structure attributes of a globular protein. A general folding/unfolding model should, therefore, be based upon structural features that transcend the peculiarities of alpha-helices, beta-sheets, and other structural motifs found in proteins. The studies presented in this paper suggest that a single structural characteristic common to all globular proteins is essential for cooperative folding. The formation of a partly folded state from the native state results in the exposure to solvent of two distinct regions: (1) the portions of the protein that are unfolded; and (2) the "complementary surfaces," located in the regions of the protein that remain folded. The cooperative character of the folding/unfolding transition is determined largely by the energetics of exposing complementary surface regions to the solvent. By definition, complementary regions are present only in partly folded states; they are absent from the native and unfolded states. An unfavorable free energy lowers the probability of partly folded states and increases the cooperativity of the transition. In this paper we present a mathematical formulation of this behavior and develop a general cooperative folding/unfolding model, termed the "complementary region" (CORE) model. This model successfully reproduces the main properties of folding/unfolding transitions without limiting the number of partly folded states accessible to the protein, thereby permitting a systematic examination of the structural and solvent conditions under which intermediates become populated. It is shown that the CORE model predicts two-state folding/unfolding behavior, even though the two-state character is not assumed in the model.

Structural energetics of the molten globule state.

Certain partly ordered protein conformations, commonly called "molten globule states," are widely believed to represent protein folding intermediates. Recent structural studies of molten globule states of different proteins have revealed features which appear to be general in scope. The emerging consensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define a general state of protein folding called "the molten globule state," which is structurally and thermodynamically distinct from both the native state and the denatured state. Despite extensive knowledge of structural features of a few molten globule states, a cogent thermodynamic argument for their stability has not yet been advanced. The prevailing opinion of the last decade was that there is little or no enthalpy difference or heat capacity difference between the molten globule state and the unfolded state. This view, however, appears to be at variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of alpha-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other potential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states.

Development and characterization of a cyclophosphamide-resistant subline of acute myeloid leukemia in the Lewis x Brown Norway hybrid rat.

Preclinical studies of resistance to alkylating agents in the Lewis x Brown Norway hybrid (LBN) rat model of acute myeloid leukemia (AML) have hitherto been limited by the sensitivity of LBN AML cells to cyclophosphamide (CY). We developed a CY-resistant subline of LBN AML by serial intravenous (IV) passage of AML cells followed by in vivo exposure to CY (100 mg/kg) 14 days later. After 18 and subsequent passages, CY-treated AML cells remained viable despite ex vivo incubation with 70 to 100 mumol/L 4-hydroperoxycyclophosphamide (4HC) or in vivo exposure to 100 to 300 mg/kg of CY. Once established, resistance to incubation with 4HC was stable in LBN AML cells after at least six serial in vivo passages without exposure to CY. Nevertheless, both control and CY-treated AML cells demonstrated similar dose-dependent sensitivity to 100 to 500 mumol/L phosphoramide mustard (PhM), the active alkylating end-product of CY activation in vivo. Levels of aldehyde dehydrogenase (ALDH), which inactivates CY by prevention of formation of PhM, were significantly elevated in these CY-resistant AML cells: cytosolic and particulate ALDH fractions from these cells were 11 to 13 times control with NAD cofactor and propanal substrate and three to four times control with NADP cofactor and benzaldehyde substrate. Further studies with this animal model of AML, in which resistance to CY is mediated by elevated ALDH activity, may elucidate mechanisms for effective elimination of drug-resistant leukemic cells ex vivo and in vivo.