Understanding radiation-induced cardiovascular damage and strategies for intervention.

There is a clear association between therapeutic doses of thoracic irradiation and an increased risk of cardiovascular disease (CVD) in cancer survivors, although these effects may take decades to become symptomatic. Long-term survivors of Hodgkin's lymphoma and childhood cancers have two-fold to more than seven-fold increased risks for late cardiac deaths after total tumour doses of 30-40 Gy, given in 2 Gy fractions, where large volumes of heart were included in the field. Increased cardiac mortality is also seen in women irradiated for breast cancer. Breast doses are generally 40-50 Gy in 2 Gy fractions, but only a small part of the heart is included in the treatment fields and mean heart doses rarely exceeded 10-15 Gy, even with older techniques. The relative risks of cardiac mortality (1.1-1.4) are consequently lower than for Hodgkin's lymphoma survivors. Some epidemiological studies show increased risks of cardiac death after accidental or environmental total body exposures to much lower radiation doses. The mechanisms whereby these cardiac effects occur are not fully understood and different mechanisms are probably involved after high therapeutic doses to the heart, or part of the heart, than after low total body exposures. These various mechanisms probably result in different cardiac pathologies, e.g. coronary artery atherosclerosis leading to myocardial infarct, versus microvascular damage and fibrosis leading to congestive heart failure. Experimental studies can help to unravel some of these mechanisms and may identify suitable strategies for managing or inhibiting CVD. In this overview, the main epidemiological and clinical evidence for radiation-induced CVD is summarised. Experimental data shedding light on some of the underlying pathologies and possible targets for intervention are also discussed.

Vascular damage as an underlying mechanism of cardiac and cerebral toxicity in irradiated cancer patients.

Radiation is an independent risk factor for cardiovascular and cerebrovascular disease in cancer patients. Modern radiotherapy techniques reduce the volume of the heart and major coronary vessels exposed to high doses, but some exposure is often unavoidable. Radiation damage to the myocardium is caused primarily by inflammatory changes in the microvasculature, leading to microthrombi and occlusion of vessels, reduced vascular density, perfusion defects and focal ischemia. This is followed by progressive myocardial cell death and fibrosis. Clinical studies also demonstrate regional perfusion defects in non-symptomatic breast cancer patients after radiotherapy. The incidence and extent of perfusion defects are related to the volume of left ventricle included in the radiation field. Irradiation of endothelial cells lining large vessels also increases expression of inflammatory molecules, leading to adhesion and transmigration of circulating monocytes. In the presence of elevated cholesterol, invading monocytes transform into activated macrophages and form fatty streaks in the intima, thereby initiating the process of atherosclerosis. Experimental studies have shown that radiation predisposes to the formation of inflammatory plaque, which is more likely to rupture and cause a fatal heart attack or stroke. This paper presents a brief overview of the current knowledge on mechanisms for development of radiation-induced cardiovascular and cerebrovascular damage. It does not represent a comprehensive review of the literature, but reference is made to several excellent recent reviews on the topic.

Lack of efficacy of Doxil in TNF-alpha-based isolated limb perfusion in sarcoma-bearing rats.

Here we show that Doxil has minimal antitumour activity in the isolated limb perfusion (ILP) setting and its activity was not enhanced by the addition of tumour necrosis factor (TNF). Doxil accumulation in tumour tissue was low and also not augmented by TNF. In contrast, activity of free conventional doxorubicin was enhanced by TNF. We conclude that application of Doxil in a TNF-based ILP is not a useful alternative to free conventional doxorubicin or melphalan.

Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis.

In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein identification and characterization of interacting sites in the same experiment. This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubulin "capping" activity of the N-terminal domain of Op18.

In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PDBEs, and polybrominated bisphenol A compounds.

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as additive flame retardants in plastics and textile materials. PBDEs are persistent compounds and have been detected in wildlife and in human adipose tissue and plasma samples. In this study, we investigated the (anti)estrogenic potencies of several PBDE congeners, three hydroxylated PBDEs (HO-PBDEs), and differently brominated bisphenol A compounds in three different cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression. In human T47D breast cancer cells stably transfected with an estrogen-responsive luciferase reporter gene construct (pEREtata-Luc), 11 PBDEs showed estrogenic potencies, with concentrations leading to 50% induction (EC(50)) varying from 2.5 to 7.3 microM. The luciferase induction of the most potent HO-PBDE [2-bromo-4-(2,4,6-tribromophenoxy)phenol] exceeded that of estradiol (E(2)), though at concentrations 50,000 times higher. As expected, brominated bisphenol A compounds with the lowest degree of bromination showed highest estrogenic potencies (EC(50) values of 0.5 microM for 3-monobromobisphenol A). In an ER alpha-specific, stably transfected human embryonic kidney cell line (293-ER alpha-Luc), the HO-PBDE 4-(2,4,6-tribromophenoxy)phenol was a highly potent estrogen with an EC(50) < 0.1 microM and a maximum 35- to 40-fold induction, which was similar to E(2). In an analogous ER beta-specific 293-ER betas-Luc cell line, the agonistic potency of the 4-(2,4,6-tribromophenoxy)phenol was much lower (maximum 50% induction compared to E(2)), but EC(50) values were comparable. These results indicate that several pure PBDE congeners, but especially HO-PBDEs and brominated bisphenol A-analogs, are agonists of both ER alpha and ER beta receptors, thus stimulating ER-mediated luciferase induction in vitro. These data also suggest that in vivo metabolism of PBDEs may produce more potent pseudoestrogens.

Towards high performance two-dimensional gel electrophoresis using ultrazoom gels.

Proteomics is the analysis of protein expression in cells or tissues, e.g., to study cellular processes at the molecular level. Ultimately, a proteome analysis should encompass most if not all protein species in a biological sample, including those present in low copy numbers. We are developing two-dimensional gel electrophoresis technology by applying narrow pH range ultrazoom gels to enhance resolution and to improve the detection of low abundance proteins. Ultrazoom gels in the acidic pH range allow the detection of proteins down to 300 copies per cell of a B-lymphoma cell line. Protein separation in the alkaline pH range, however, still requires optimization, especially in conjunction with high sample loads.

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain.

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.

A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification.

We describe a method for generating multiple small sequences from the N terminal of peptides in unseparated protein digests by stepwise thioacetylation and acid cleavage. The mass differences between a series of N-terminally degraded peptides give short sequences of defined length. Such short "sequence tags" together with the mass of the parent peptide can be used to identify the protein in a database. The sequence ladders are generated without the use of chain terminators or sample aliquoting and the degradation reagents are water soluble so that the chemistry can be carried out on peptides immobilized on C-18 reversed-phase supports without any peptide loss due to washing with organic solvents as occurs in Edman type sequencing. The entire procedure can be automated, and we describe a prototype device for the parallel analysis of multiple samples. We demonstrate the effectiveness of this chemical tagging method in a comparison with Edman sequencing, peptide mass fingerprinting, and MS/MS analysis of crude protein fractions obtained from an HPLC separation of the Escherichia coli ribosome complex which consists of 57 proteins. We show that chemical tagging is a viable first-pass high-throughput identification method to be used prior to an in depth MS/MS analysis.

An algorithm for the identification of proteins using peptides with ragged N- or C-termini generated by sequential endo- and exopeptidase digestions.

We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, 'Tag searching', in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, 'Rag Tag' searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation. The mixture is analyzed by matrix assisted laser desorption and ionization time of flight mass spectrometry and a list of intact peptide masses is generated, each associated with a set of degradation product masses which serve as unique tags. These 'tagged masses' are used as the input to an algorithm we have written, MassDynSearch, which searches protein and DNA databases for proteins which contain similar tagged motifs. The method is simple, rapid and can be fully automated. The main advantage of this approach is that the specificity of the initial digestion is unimportant since multiple peptides with tags are used to search the database. This is especially useful for proteins like membrane, cytoskeletal, and other proteins where specific endopeptidases are less efficient and lower specificity proteases such as chymotrypsin, pepsin, and elastase must be used.

Sample handling for proteome analysis.

The main factor limiting the sensitivity range for the identification of proteins isolated by two-dimensional (2-D) electrophoresis is sample handling: protein detection limits on the gel, losses during extraction and digestion, as well as interference of gel contaminants and detergents with the mass spectrometry (MS) detection increasing background noise. At the one hundred picomole level, losses are fairly negligible but when the amounts drop below 1 picomole (and subfemtomole peptide detection limits have been reported recently by MS), the losses become a critical point. In order to extend proteome analysis to include very low copy number proteins, methods must be developed to minimize losses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we have developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)-free form which does not require staining, (ii) increase protein extraction and digestion yields and lower the contamination by autoproteolytic products, and (iii) allow direct modification of the peptide mixture to generate sequence tags.

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli.

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a sigma N (sigma 54) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.

Novel aromatic isothiouronium derivatives which act as high affinity competitive antagonists of alkali metal cations on Na/K-ATPase.

This paper describes properties of a novel family of aromatic isothiouronium derivatives, which act as Na(+)-like competitive antagonists on renal Na/K-ATPase. The derivatives are reversible competitors of Rb+ and Na+ occlusion. Ki values of the most potent compounds, 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) and 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene(Br2-TITU ), 0.65 and 0.32 microM, respectively, are 15-30-fold lower than Ki values of the bis-guanidinium derivatives described previously (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149), and represent the lowest reported values for cation antagonists. Using fluorescein-labeled Na/K-ATPase, all derivatives have been shown to stabilize the E1 conformation when bound at high affinity sites (i.e. they are sodium-like). In addition, in one condition (10 mM Tris-HCl, pH 8.1), high concentrations of Br-TITU (KD approximately 10 microM) appear to stabilize an E2 conformation. We propose a model which allows for simultaneous binding of the antagonists to high affinity cytoplasmic sites and low affinity sites, which may be at the extracellular surface. Blockage of cation occlusion by the isothiouronium derivatives at the cytoplasmic surface probably occurs at the entrance to the occlusion sites, which is recognized both by Na+ antagonists and by Na+ or K+ ions. Unlike the alkali metal cations, the Na+ antagonists are not occluded or transported (see also Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). The isothiouronium derivatives appear to be promising candidates for further development as affinity labels of cation binding domains, for kinetic analysis of isoforms or mutated Na/K pumps, or as probes of other cation transport proteins.

An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade.

The PII protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a PII homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli PII. It contains the conserved tyrosine residue which is known to be the site of uridylylation in PII. E. coli is the first organism in which two different PII proteins have been detected.

Energy, control and DNA structure in the living cell.

Maintenance (let alone growth) of the highly ordered living cell is only possible through the continuous input of free energy. Coupling of energetically downhill processes (such as catabolic reactions) to uphill processes is essential to provide this free energy and is catalyzed by enzymes either directly or via "storage" in an intermediate high energy form, i.e., high ATP/ADP ratio or H+ ion gradient. Although maintenance of a sufficiently high ATP/ADP ratio is essential to overcome the thermodynamic burden of uphill processes, it is not clear to what degree enzymes that control this ratio also control cell physiology. Indeed, in the living cell homeostatic control mechanisms might exist for the free-energy transduction pathways so as to prevent perturbation of cellular function when the Gibbs energy supply is compromised. This presentation addresses the extent to which the intracellular ATP level is involved in the control of cell physiology, how the elaborate control of cell function may be analyzed theoretically and quantitatively, and if this can be utilized selectively to affect certain cell types.

Extensive digestion of Na+,K(+)-ATPase by specific and nonspecific proteases with preservation of cation occlusion sites.

This paper extends our recent report that renal Na+,K(+)-ATPase is digested by trypsin in the absence of Ca2+ and presence of Rb+ ions to a stable 19-kDa fragment and smaller membrane-embedded fragments of the alpha chain and essentially intact beta chain. These are referred to as "19-kDa membranes." Occlusion of both Rb+ (K+) or Na+ ions is preserved, but ATP-dependent functions are lost (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570). We now show that extensive digestion with nonselective fungal proteases (Pronase and proteinase K) alone, in combination, or after tryptic digestion can remove up to 70% of membrane protein without destroying Rb+ occlusion. In the most heavily digested membranes, the 19-kDa fragment or a slightly shorter 18.5-kDa fragment and smaller fragments of the alpha chain remain, whereas the beta chain is largely digested, leaving smaller membrane-embedded fragments (13-15 kDa). For either trypsin or Pronase digestion, preservation of Rb+ occlusion and the specific fragmentation pattern is observed only in the absence of divalent metal ions (Mg2+ or Ca2+) and presence of either Rb+ or Na+ or congener ions. Tryptic digestion at pH 7.0 can split the beta chain into two fragments of approximately 50 and 16 kDa joined by an S-S bridge. The 16-kDa fragment is protected against further digestion by the presence of Rb+ ions, but probably is not directly involved in occluding cations. Tryptic 19-kDa membranes show a clear and reproducible fragmentation pattern in which all predicted membrane segments are identifiable. Families of fragments from 19-kDa membranes, including seven peptides of 7.6-11.7 kDa, have been separated by size-exclusion high performance liquid chromatography, concentrated, and resolved on 16.5% Tricine gels. N-terminal sequences of the different fragments have been determined after transfer to polyvinylidene difluoride paper. The most interesting findings are as follows. (a) Whereas the 19-kDa tryptic fragment begins at Asn831 as reported previously, the 18.5-kDa Pronase fragment begins at Thr834. (b) Fragments in tryptic 19-kDa membranes of 7.6-11.7 kDa begin at Asp68, Ile263, and Gln737, respectively. These include all putative transmembrane segments other than those in the 19-kDa fragment. (c) A Pronase fragment of 7.8 kDa begins at Thr834, i.e. apparently the 19-kDa fragment has been partially cut, without loss of Rb+ occlusion. (d) Tryptic 16- and approximately 50-kDa fragments of the beta chain begin at Ala5 and Gly143, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the cation binding domain of Na/K-ATPase.

This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.

Effect of free fatty acids and detergents on H,K-ATPase. The steady-state ATP phosphorylation level and the orientation of the enzyme in membrane preparations.

The effects of detergents and free fatty acids on the K(+)-activated ATPase activity and on the steady-state phosphorylation level of pig gastric H,K-ATPase were studied. Unsaturated free fatty acids inhibited the K(+)-activated ATPase activity, due to inactivation of the enzyme (long-term effects) and to a decrease in the K(+)-sensitive dephosphorylation rate (short-term effects). The degree of inhibition depended on the reaction conditions: the protein concentration, the temperature and the ligands used. No effect was observed when saturated- or methylated unsaturated fatty acids were tested. Free fatty acids and the detergent C12E8 increased the steady-state ATP phosphorylation level, indicating the presence of vesicular structures in the H,K-ATPase preparations. At higher concentrations these compounds inactivated H,K-ATPase, which was measured as a decrease in phosphorylation capacity. By combining the data from the ATP phosphorylation level in the absence and presence of C12E8 (without inactivation) and the data from the K(+)-activated ATPase activity with and without ionophore the tightness of vesicular preparations and the orientation of H,K-ATPase was determined. A rather simple method for the isolation of H,K-ATPase is reported, which yields highly purified H,K-ATPase preparations with a ATP phosphorylation capacity of 3.9 nmol P per mg protein or 0.57 mol P per mol alpha beta protomer. This number suggests that each alpha-subunit H,K-ATPase can be phosphorylated at the same time.