Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.


X-ray analysis of substrate analogs in the ricin A-chain active site.

Ricin A-chain is an N-glycosidase that hydrolyzes the adenine ring from a specific adenosine of rRNA. Formycin monophosphate (FMP) and adenyl(3'-->5')guanosine (ApG) were bound to ricin A-chain and their structures elucidated by X-ray crystallography. The formycin ring stacks between tyrosines 80 and 123 and at least four hydrogen bonds are made to the adenine moiety. A residue invariant in this enzyme class, Arg180, appears to hydrogen bond to N-3 of the susceptible adenine. Three hypothetical models for binding a true hexanucleotide substrate, CGAGAG, are proposed. They incorporate adenine binding, shown by crystallography, but also include geometry likely to favor catalysis. For example, efforts have been made to orient the ribose ring in a way that allows solvent attack and oxycarbonium stabilization by the enzyme. The favored model is a simple perturbation of the tetraloop structure determined by nuclear magnetic resonance for similar polynucleotides. The model is attractive in that specific roles are defined for conserved protein residues. A mechanism of action is proposed. It invokes oxycarbonium ion stabilization on ribose by Glu177 in the transition state. Arg180 stabilizes anion development on the leaving adenine by protonation at N-3 and may activate a trapped water molecule that is the ultimate nucleophile in the depurination.


Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.


Domain structure and domain-domain interactions in the carboxy-terminal heparin binding region of fibronectin.

The domain structures and stabilities of fragments isolated from the so-called 'hep 2' region of plasma fibronectin have been investigated by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The 30 kDa hep-2A fragment contains three type III modules (III12 to III14), whereas the 40 kDa hep-2B fragment contains four such modules (III12 to III15). Melting of these fragments at neutral pH was irreversible and accompanied by rapid aggregation. In contrast, melting was completely reversible in 50 mM-glycine at pH 2.7, where DSC measurements revealed the presence of three independently folded domains in 30kDa hep-2A and four in 40 kDa hep-2B. That each domain represented a single module was confirmed by measurements with four single-module subfragments, all of which melted reversibly, even at neutral pH. At neutral pH in the presence of 6 M-urea, 30 kDa hep-2A melted reversibly in a sharp peak from which only two transitions could be resolved by deconvolution. Only the larger of these was stabilized by heparin and was assigned to modules III13 and III14. Upon isolation, module III13 melted at lower temperature than in the parent fragment where it is stabilized through an interaction with module III14. We conclude that all type III modules in the hep-2 region of fibronectin constitute independently folded domains. Modules III13 and III14 form a highly co-operative structure through functionally significant interactions that can be disrupted with acid or sufficient concentrations of urea or guanidinium chloride.


Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.


Binding of the dye congo red to the amyloid protein pig insulin reveals a novel homology amongst amyloid-forming peptide sequences.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.


Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.


Stability of a model beta-sheet in water.

We have used molecular dynamics simulations to determine the stability in water of a model beta-sheet formed by two alanine dipeptide molecules with two intermolecular hydrogen bonds in the closely spaced antiparallel arrangement. In this paper we describe our computations of the binding free energy of the model sheet and a portion of the free energy surface as a function of a reaction co-ordinate for sheet formation. We used the free energy surface to identify stable conformations along the reaction co-ordinate. To determine whether or not the model sheet with two hydrogen bonds is more stable than a single amide hydrogen bond in water, we compared the results of the present calculations to results from our earlier study of linear hydrogen bond formation between two formamide molecules (the formamide "dimer"). The free energy surfaces for the sheet and formamide dimer each have two minima corresponding to locally stable hydrogen-bonded and solvent-separated configurations. The binding free energies of the model sheet and the formamide dimer are -5.5 and -0.34 kcal/mol, respectively. Thus, the model sheet with two hydrogen bonds is quite stable while the simple amide hydrogen bond is only marginally stable. To understand the relative stabilities of the model sheet and formamide dimer in terms of solute-solute and solute-water interactions, we decomposed the free energy differences between hydrogen-bonded and solvent-separated conformations into energetic and entropic contributions. The changes in the peptide-peptide energy and the entropy are roughly twice as large for the sheet as they are for the formamide dimer. The magnitude of the peptide-water energy difference for the sheet is less than twice (by about 3.5 kcal/mol) that for the formamide dimer, and this accounts for the stability of the sheet. The presence of the side-chains and/or blocking groups apparently prevents the amide groups in the sheet from being solvated as favorably in the separated arrangement as in the formamide dimer, where the amide groups are completely exposed to the solvent.


Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution. The space group is P2(1)2(1)2(1) (a = 64.5 A, b = 70.5 A and c = 120.1 A), with one Fab-protein G domain complex in the asymmetric unit. Solution of the three-dimensional structure of the complex will permit a detailed analysis of the molecular interactions between protein G and the Fab portion of IgG.


Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.


Purification, crystallization and preliminary X-ray analysis of the 3-phosphoglycerate kinase from Bacillus stearothermophilus.

As part of a programme investigating the molecular basis of thermal stability in proteins we have isolated and characterized the thermally stable 3-phosphoglycerate kinase (PGK) from Bacillus stearothermophilus NCA 1503. The B. stearothermophilus PGK has been crystallized in a form suitable for X-ray diffraction analysis. Crystals which diffract to greater than 1.8 A resolution have been grown in the presence of the nucleotide substrate, MgATP, using polyethylene glycol (PEG 600) as a precipitant. The best crystals have been obtained using "seeding" techniques and are monoclinic, space group P2(1), with cell dimensions a = 40.5 A, b = 74.0 A, c = 68.5 A and beta = 99.8 degrees.


Crystallization and initial crystallographic results for pepstatin A inhibited bovine cathepsin D.

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5.9. The crystals diffract to a resolution of 3.0 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 74.8 A, b = 76.0 A, c = 157.7 A. There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0.014 A and 3.2 degrees, respectively, and a crystallographic R factor of 17%.


Complete nucleotide sequence and structural organization of rat cardiac troponin T gene. A single gene generates embryonic and adult isoforms via developmentally regulated alternative splicing.

We have previously demonstrated that rat cardiac troponin T (TnT) is expressed as two different isoforms during development, the larger, more acidic embryonic isoform and the smaller, more basic adult isoform, which appear to be generated from a common transcript of the cardiac TnT gene by alternative RNA splicing. In this study, Southern blot analysis confirmed the existence of a single copy of cardiac TnT gene in the rat genome. For investigation of the molecular mechanism of isoform switch and the control of this gene expression in myocardial development, several overlapping genomic clones were isolated from a rat genomic library. Complete nucleotide sequences were determined from these genomic clones and revealed a 19,186 base-pair DNA fragment containing 16 exons of rat cardiac TnT gene. Its DNA sequence and exon organization appeared to differ from that of the rat fast skeletal muscle TnT gene or chicken cardiac TnT gene. Comparison of genomic and cDNA clones also confirmed that the cardiac TnT isoform switching was due to the inclusion or exclusion of exon 4 during RNA processing. Sequence analysis allowed us to further identify the other alternatively spliced exon containing only nine nucleotides in size (exon 12). The inclusion and complete or partial exclusion of this exon may be responsible for generating three classes of mRNAs detected by our cDNA clones. The functional significance of this variation in TnT isoforms remained unknown, but its splicing pattern did not appear to link to the developmental changes. The 5' upstream structure was very similar to that in chicken cardiac TnT gene but differed from that in the rat fast skeletal muscle TnT gene, suggesting a similar regulatory mechanism for mammalian and avian cardiac TnT expression.


Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.

Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.


Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the alpha-subunit of RNA polymerase.

Escherichia coli integration host factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the alpha subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.


Amino acid preferences of small proteins. Implications for protein stability and evolution.

The dependence of amino acid frequency on sequence length has been examined for the 20 natural amino acids using a set of 2275 protein sequences with little sequence identity. As expected, the frequency of cysteine increases dramatically for sequences shorter than 100 amino acids with a length-dependence that corresponds to an average of two Cys per sequence independent of length. Surprisingly dramatic changes were also observed for the frequencies of arginine, lysine, aspartic acid, and glutamic acid: Arg and Lys frequencies increase for short sequences whereas Asp and Glu frequencies decrease. These changes do not appear to be due to an over-abundance of DNA- and membrane-binding proteins in the database and may, therefore, be related to protein stability. Possible stabilizing mechanisms include increased hydrogen bonding by Arg and increased hydrophobic stabilization due to the amphiphilic character of Arg and Lys. These observations suggest that amino acid composition played an important role in the evolution of small proteins.


Positive and negative effects of DNA bending on activation of transcription from a distant site.

Transcription of the Escherichia coli glnHPQ operon, which encodes components of the high-affinity glutamine transport system, is activated by nitrogen regulator I (NRI)-phosphate in response to nitrogen limitation. NRI-phosphate binds to sites upstream from the sigma 54-dependent glnHp2 promoter and activates transcription by catalyzing the isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. On linear DNA, the initiation of glnHp2 transcription requires in addition to NRI-phosphate the presence of integration host factor (IHF), which binds to a site located between the NRI-binding sites and the promoter. On supercoiled DNA, IHF does not play an essential role, but enhances the activation of transcription by NRI-phosphate. We found that at a mutant glnHp2 promoter with increased affinity for sigma 54-RNA polymerase, the initiation of transcription can be activated equally well by NRI-phosphate in the presence or absence of IHF. Binding of IHF to its site does not increase the binding of sigma 54-RNA polymerase to the glnHp2 promoter; instead, our data suggest that IHF bends the DNA to align the activator with the closed sigma 54-RNA polymerase promoter complex to facilitate the interactions that result in open complex formation. In the absence of IHF, NRI-phosphate can activate transcription whether its binding sites are on the same face of the DNA helix as the sigma 54-RNA polymerase or on the opposite face. IHF enhances transcription when the three proteins are located on the same face of the helix, but strongly inhibits transcription when any one of the proteins is located on the opposite face.


Skeletal development in Xenopus laevis (Anura: Pipidae).

Postembryonic skeletal development of the pipid frog Xenopus laevis is described from cleared-and-stained whole-mount specimens and sectioned material representing Nieuwkoop and Faber developmental Stages 46-65, plus postmetamorphic individuals up to 6 months old. An assessment of variation of skeletogenesis within a single population of larvae and comparison with earlier studies revealed that the timing, but not the sequence, of skeletal development in X. laevis is more variable than previously reported and poorly correlated with the development of external morphology. Examination of chondrocranial development indicates that the rostral cartilages of X. laevis are homologous with the suprarostral cartilages of non-pipoid anurans, and suggests that the peculiar chondrocranium of this taxon is derived from a more generalized pattern typical of non-pipoid frogs. Derived features of skeletal development not previously reported for X. laevis include 1) bipartite formation of the palatoquadrate; 2) precocious formation of the adult mandible; 3) origin of the angulosplenial from two centers of ossification; 4) complete erosion of the orbital cartilage during the later stages of metamorphosis; 5) development of the sphenethmoid as a membrane, rather than an endochondral bone; and 6) a pattern of timing of ossification that more closely coincides with that of the pelobatid frog Spea than that recorded for neobatrachian species.


The ear in subterranean insectivora and rodentia in comparison with ground-dwelling representatives. I. Sound conducting system of the middle ear.

Compared to acoustically unspecialized mammals (soricids and murids), the middle ear of subterranean insectivores and rodents (twelve species of six families examined) was clearly distinguished and characterized by many common features: rather round and relatively larger eardrum without a pars flaccida; reduced gonial; loose or no connection between the malleus and the tympanic bone; reduced and straightened transversal part of the malleus; enlarged incus; increased and rather flat incudo-mallear joint; rather parallel position of the mallear manubrium and incudal crus longum in some species (and their fusion in bathyergids); reduced or even missing middle ear muscles. Convergent occurrence of these structural features in taxa of different origin and their generally derived character suggest that they cannot be categorized as degenerative. The form of the stapes can be considered as a non-adaptive trait; it was taxon specific yet remarkably polymorphous in some species and exhibited no convergent features among subterranean mammals. Structural retrogression resulting in a columella-like stapes was observed in some species lacking the stapedial artery. The stapedial base was relatively larger than in unspecialized mammals. The subterranean mammals did not exhibit conspicuously enlarged eardrums as would be required for sensitive tuning to low frequencies. It is, however, argued that while selective pressures in the subterranean ecotope promoted hearing of low frequencies, hearing sensitivity did not have to be enhanced.


The functional morphology of xenarthrous vertebrae in the armadillo Dasypus novemcinctus (Mammalia, Xenarthra).

In order to assess the mechanical properties of xenarthrous vertebrae, and to evaluate the role of xenarthrae as fossorial adaptations, in vitro bending tests were performed on posterior thoracic and lumbar vertebral segments excised from specimens of the armadillo Dasypus novemcinctus and the opossum Didelphis virginiana, the latter being used to represent the primitive mammalian condition. The columns of the two species were subjected to dorsal, ventral, and lateral bending, as well as torsion, in order to determine their stiffness in each of these directions. During these tests, bone strains in the centra of selected vertebrae were determined using rosette strain gages. Overall stiffness of the armadillo backbone at physiologically relevant displacement levels was significantly higher than that of the opossum for both dorsal and lateral bending. The two species also exhibited significant differences in angular displacement of individual vertebrae and in vertebral strain magnitudes and orientations in these two directions. No significant differences were observed when the columns of the two species were subjected to torsion or to ventral bending. Our results suggest that some, but not all, of the mechanical differences between the two species are due to the presence of xenarthrae. For example, removal of the xenarthrae from selected vertebrae (L2-L4) changes strain orientation and shear, but not strain magnitudes. Comparisons with functional data from other digging mammals indicate that the modified mechanical properties of the Dasypus column are consistent with an interpretation of xenarthrae as digging adaptations and lend support to the idea that the order Xenarthra represents an early offshoot of placental mammals specialized for fossoriality.


Ultrastructural changes in guinea pig endometrial cells during the estrous cycle.

In the guinea pig, the estrous cycle is characterized by a constant measurable level of plasma progesterone with two peaks: the first one associated with the peak of plasma estradiol-17 beta occurring at proestrus and the second, during diestrus, more pronounced at the time at which the level of estradiol-17 beta is undetectable. The progesterone receptor content is the highest on day 1 and the lowest on day 10 of the estrous cycle, which lasts 16.3 +/- 1.5 days (n = 37; mean +/- SD). There is a positive correlation between the plasma level of estradiol-17 beta and the progesterone receptors detected immunocytochemically in both endometrial epithelial and stromal cells. The general morphology of the endometrium during proestrus and estrus is consistent with an estrogenic stimulation, i.e., a smooth and regular surface of the endometrium and the presence of numerous microvilli on the cell surface. However, a moderate secretory activity also occurs in proestrus and estrus. During postestrus, the glandular cells display an increase in characteristic secretory features which parallels the rise of progesterone in the plasma.


Myoglobin supported oxygen consumption in isolated rat hearts under dysoxic conditions.

Oxygen consumption was assessed in contracting, isolated rat hearts subjected to Langendorff perfusion. Initially, hearts were perfused with Krebs-Henseleit bicarbonate medium (KHB). Some hearts were treated with a 10 min pulse of medium containing 0.05 mM phenylhydrazine to oxidize approximately 78% of myoglobin to a state incapable of binding oxygen. Stepwise reduction in input PO2 resulted in a decline in oxygen consumption (MO2) in control and treated hearts. Phenylhydrazine treatment had no effect upon MO2 in hearts perfused with medium having a PO2 of about 585 mmHg or higher. However, at an input PO2 of approximately 370 mmHg, MO2 was decreased to 60% of the level at an input PO2 of 710 mmHg in untreated hearts and significantly lower to 32% of initial level in myoglobin blocked hearts. In subsequent experiments, hearts were perfused with KHB containing human red blood cells (RBCs) to elevate the oxygen content of the perfusate. The addition of RBCs to medium having a PO2 of approximately 140 mmHg resulted in enhancement of MO2 and maintenance of performance. But the preparations were considered to be dysoxic since MO2 with RBCs in the medium (PO2 approximately 140 mmHg) was lower than under perfusion with KHB (PO2 approximately 710 mmHg). This should however not detract from the utility of the model in elucidating myoglobin function under oxygen limiting conditions. At an input PO2 of 140 mmHg hearts treated with phenylhydrazine to impede myoglobin function had a significantly lower MO2 and viability than untreated hearts.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison between the effects of 2-3 butanedione monoxime (BDM) and calcium chloride on myocardial oxygen consumption.

The agent 2,3-butanedione monoxime (BDM) has been reported to reduce the sensitivity of myofilament force development to calcium ions, without affecting the calcium transient in myocardium. One would predict, therefore, that BDM should reduce the contractile state of the heart without reducing the amount of oxygen that is consumed to fuel the process of excitation-contraction coupling. The purpose of the present experiment was to test this hypothesis using isovolumically contracting, isolated, blood perfused canine hearts during beta-blockade induced by continuous intra-coronary infusion of propranolol (1 mg/h). Contractile state was increased in seven hearts by CaCl2 infusion. Subsequently, while the CaCl2 infusion was continued at the highest rate, contractile state was reduced by BDM infusion. At each contractile state, we measured the left-ventricular end-systolic pressure-volume relation (ESPVR), the relation between myocardial oxygen consumption and its mechanical correlate, pressure-volume area (MVO2 vs PVA), and the duration of the LV pressure waveform. Contractile state was quantified by interpolated developed pressure at a reference ventricular volume of 25 ml (P25). BDM infusion (0.5-7 mM) caused a dose-dependent reduction in contractile state (50% reduction in P25 at 2.4 +/- 0.3 mM), and a dose-independent increase in coronary blood flow. Furthermore, BDM significantly reduced the duration of the pressure waveform up to 40% at the highest rate of BDM infusion compared to the pressure waveform duration measured at maximum CaCl2 infusion. We observed a direct relationship between MVO2 of the mechanically unloaded heart and contractility; this relation was unaffected by BDM infusion (P > 0.3). The slope of the MVO2-PVA relation decreased with increases in contractile state, but this decrease was unaffected by BDM (P > or = 0.4). We conclude that in the isolated canine heart, BDM does not act energetically as expected for a myofibrillar calcium desensitizing agent.


Effect of food restriction on the phosphocreatine energy shuttle components in rat heart.

Malnutrition has been associated with changes in cardiac metabolism and performance. We have previously reported a diabetic-type cardiomyopathy associated with chronic food restriction and weight loss. Because the creatine-phosphocreatine-creatine kinase system is important in the contractile process, we studied the components of this system in rats fed a food-restricted diet (33% of control animal intake). After 4 weeks of food restriction, total creatine kinase (CK) activities were reduced by 28% in ventricles and by 38% in atria. The CK isoenzymes in the heart were not equally affected. The BB isoenzyme was decreased by 77% and 78%, the MB isoenzyme by 45% and 43%, the MM isoenzyme by 22% and 19% and CKmito by 16% and 15% in ventricles and atria, respectively. In contrast, brain CK activity which is predominantly the BB isoenzyme, was slightly higher in the food-restricted than in control rats. Further studies on ventricular tissue from food-restricted rats revealed a 27% decline in myofibrillar CR activity and a 58% decline in myofibrillar ATPase activity. Phosphocreatine and creatine concentrations were not changed by food restriction, however, ATP was decreased by 23% in ventricles from rats on the restricted diet. Cardiac mitochondrial oxidative phosphorylation was also impaired. State 3 respiration with alpha-ketoglutarate was reduced 20% in the food-restricted heart. These changes are compared to those which we previously observed in the diabetic rat heart and the significance of these findings is discussed.


Oxidative metabolism and mechanical function in reperfused neonatal pig heart.

Oxidative metabolism in reperfused neonatal myocardium has not been characterized. A blood-perfused isovolumic heart preparation was used to quantify metabolic and mechanical responses of the neonatal left ventricle to global normothermic ischemia and reperfusion. Hearts from piglets aged 2-7 days were subjected to either 2 hrs of total ischemia at 37 degrees C followed by 1 hr of reperfusion or 3 hrs of perfusion alone; glucose and palmitate oxidation were measured in separate experiments by incorporation of the appropriate [14C]-labeled substrate into the perfusate. In the pre-ischemic period, glucose, palmitate, and lactate contributed 10%, 41%, and 36%, respectively, to oxidative metabolism. After 2 hrs of total normothermic ischemia, oxidation of exogenous glucose was 165% and 229% of control values at 30 and 60 minutes of reperfusion, respectively; palmitate oxidation was 110% and 143% of control values at these times. Despite increased glucose oxidation, palmitate oxidation accounted for 69% of myocardial oxygen consumption after 1 hr of reperfusion, with glucose responsible for 25%. Lactate use was minimal during reperfusion. Reperfusion was accompanied by rapid and parallel recovery of oxygen utilization, mechanical function, and high-energy phosphates. The neonatal piglet heart demonstrates significant metabolic and mechanical tolerance to prolonged ischemia. Although glucose utilization increased markedly, palmitate was the primary substrate for energy production in the post-ischemic neonatal heart.


Action potentials and potassium currents in rat ventricular muscle during experimental diabetes.

Time course of the surface electrical activity was studied in left ventricular trabeculae of Wistar rats made diabetic using streptozotocin. The action potentials were recorded in Tyrode's solution at 32 degrees C, their duration considerably increased in diabetes. By the 8th week, the prolongation was 64% at 25% of repolarization; 112% at 50% and 118% at 75%. Insulin treatment reduced the prolongation of the action potentials although a complete restoration was not achieved. 0.1 mM La3+ moderately shortened the electrical activity both in control and in diabetic trabeculae. Three mM 4-aminopyridine made the time course of control action potentials very similar to the diabetic ones while the action potentials from the diabetic animals were prolonged further to a smaller extent. Whole-cell clamp experiments in isolated ventricular myocytes (20-23 degrees C) showed a considerable decrease and a somewhat accelerated inactivation of the transient outward current (Ito) in diabetes. The steady-state inactivation and the rate of recovery from inactivation of Ito did not change. No alterations in the magnitude and voltage dependence of inward rectifier (IK1) were found around the resting membrane potential. The diabetes-related suppression of Ito explains the decreased repolarization rate of action potentials.


Triacylglycerol metabolism in hypoxic, glucose-deprived rat cardiomyocytes.

We have recently shown that a triacylglycerol (TG)-fatty acid cycle is operating in rat myocardial cells incubated in a hypoxic, glucose-containing incubation medium (Myrmel et al., 1991a). In the present study we investigated whether this cycle occurred in hypoxic, glucose-deprived myocytes, and whether high TG levels would increase TG-fatty acid cycling and thereby energy consumption. Myocytes with elevated contents of TG were obtained from the hearts of streptozotocin-induced diabetic rats (diabetic myocytes) and from normal rat myocytes prepared in the presence of oleic acid (TG-loaded myocytes). The TG content of diabetic and TG-loaded myocytes prior to hypoxic incubations was more than two times higher (P < 0.05) than that of their respective controls (123.8 +/- 20.6 and 125.3 +/- 12.7 vs 56.8 +/- 6.0 and 58.6 +/- 9.4 nmol/10(6) cells, mean +/- S.E., n = 7). Only diabetic and TG-loaded myocytes expressed marked reductions in TG content during glucose free incubations. There were no differences in TG-fatty acid cycling between the various myocyte groups, calculated as the difference between glycerol output and the concomitant decrease in TG (range: 36.7 +/- 8.1- 48.9 +/- 9.7 nmol TG/10(6) cells.2h). Apparently, the cycle was continuous throughout the whole incubation period despite falling ATP levels, contracture (rounding up) of myocytes, as well as cessation of glycogenolysis after about 40 min incubation. The cellular content of glycerol-3-phosphate, known to control TG-fatty acid cycling, increased continuously and to the same extent throughout the 2 h incubation period. Futile energy consumption associated with TG-fatty acid cycling, amounted to approximately 30% of total cellular energy consumption for the whole incubation period. In conclusion, hypoxic glucose deprived rat myocytes show TG-fatty-acid cycling, even after cessation of glycogenolysis. The extent of cycling, and thus the energy cost associated with it, was not influenced by the initial level of TG in the myocytes. We propose that glycerol-3-phosphate needed to fuel the TG-fatty acid cycle after exhaustion of the glycolytic supply is derived from phospholipid degradation.


The protective role of heat stress in the ischaemic and reperfused rabbit myocardium.

Cells subjected to increases in temperature induce the expression of several proteins known as heat shock or stress proteins. This process enhances the cell's ability to overcome the effects of further stress. In this respect, the effects of heat stress have been reported to protect the hearts of rats following ischaemia and reperfusion. We have confirmed and extended this observation, not only using different indices of myocardial injury but also in another species, namely the rabbit. Animals were anaesthetized and the body temperature raised to 42 degrees C for a 15-min period. Controls were treated in the same way but without heating. Twenty-four hours later the rabbits were re-anaesthetized and the hearts removed for either heat stress protein analysis or perfusion with Krebs buffer using an isolated perfused heart apparatus. Hearts were subjected to 60 min of low flow (1 ml/min) ischaemia followed by 30 min of reperfusion. All hearts subjected to heat stress showed an enhanced recovery of function upon reperfusion as measured by improvements in developed pressure (27.3 +/- 3.6 vs 16.3 +/- 3.0 mmHg) and diastolic pressure (37.3 +/- 7.4 vs 54.7 +/- 3.1 mmHg). In addition, creatine kinase release, associated with reperfusion, was significantly reduced in the heat-stressed hearts (532 +/- 102 vs 1138 +/- 73 mU/min/g wet wt). Myocardial accumulation and release of oxidized glutathione, an index of oxidative stress, was significantly reduced in the heat-stressed group (0.003 +/- 0.003 vs 0.376 +/- 0.113 nmol/min/g wet wt). The improved metabolic status of the reperfused heat-stressed hearts was further demonstrated by a significant conservation in the levels of ATP (6.1 +/- 0.9 vs 2.8 +/- 0.8 mumol/g dry wt) and CP (36.9 +/- 6.4 vs 16.4 +/- 5.1 mumol/g dry wt). Finally, isolated mitochondrial function in terms of respiratory control index (RCI) was maintained in the heat-stressed hearts (9.2 +/- 0.9 vs 5.7 +/- 0.2) and overloading with calcium was reduced. These data extend the hypothesis that heat stress protects the heart following ischaemia and reperfusion in this in vitro model, in a way as yet undetermined.


Effects of duration of ischaemia during preconditioning on mechanical function, enzyme release and energy production in the isolated working rat heart.

Repeated brief episodes of ischaemia "precondition" the myocardium and protect it during a subsequent period of sustained ischaemia. We subjected isolated rat hearts to sustained ischaemia with or without reperfusion after different schedules of preconditioning. We demonstrated that preconditioning with three 5 min periods of ischaemia separated by 10 min periods of reperfusion permits better functional recovery than preconditioning with three 2 min ischaemic periods separated by 10 min of reperfusion. Preconditioned hearts had creatine phosphate and adenine nucleotide levels comparable to those in the aerobically perfused controls, and showed good functional recovery. Although the mechanisms by which preconditioning protects the heart from subsequent ischaemic damage are unclear, we speculate that preservation of mitochondrial function and oxidative energy production is involved.


PEG-SOD and myocardial antioxidant status during ischaemia and reperfusion: dose-response studies in the isolated blood perfused rabbit heart.

We have previously shown that the polyethylene glycol conjugated superoxide dismutase (SOD), which has a plasma half-life of more than 24 h, protects the blood perfused rabbit heart against injury during ischaemia and reperfusion. However, the profile for the dose-dependency of protection was bell-shaped with loss of efficacy below 6000 and above 30,000 U/kg. In the present study, isolated rabbit hearts, perfused with blood from support rabbits, were subjected to a 2 min infusion with St Thomas' Hospital cardioplegic solution followed by 60 min of global ischaemia (37 degrees C) and 60 min of reperfusion. PEG-SOD was administered 1 h or 12-24 h before ischaemia. We assessed the effect of PEG-SOD on ischaemia- and reperfusion-induced changes in: (i) the tissue content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) and (ii) the activity of CuZn-SOD, Mn-SOD and glutathione peroxidase and reductase (GPD and GRD). Ischaemia and reperfusion reduced tissue GSH content by 70% and increased GSSG content by 400% (from their fresh aerobic values of 13.1.9 and 0.09 +/- 0.01 nmol/mg protein, respectively). PEG-SOD, given intravenously at various doses to donor and support rabbits 1 h or 12-24 h before ischaemia, protected against these changes with a bell-shaped dose-response relationship. Thus, with 0, 3000, 6000, 12,000, 30,000 and 60,000 U/kg, GSH content was 4.1 +/- 0.4, 4.8 +/- 0.4, 8.5 +/- 0.5, 12.3 +/- 1.6, 12.3 +/- 1.6 and 5.0 +/- 0.5 nmol/mg protein in the 1 h pretreatment group and 4.1 +/- 0.4, 4.2 +/- 0.5, 10.4 +/- 1.5, 11.2 +/- 1.1, 11.4 +/- 0.7 and 4.7 +/- 0.6 nmol/mg protein in the 12-24 h pretreatment group (means +/- S.E.M.). For GSSG the corresponding values were 0.36 +/- 0.04, 0.34 +/- 0.03, 0.12 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.41 +/- 0.03 nmol/mg protein for the 1 h group and 0.36 +/- 0.04, 0.35 +/- 0.02, 0.15 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.34 +/- 0.02 nmol/mg protein for the 12-24 h group. Ischaemia and reperfusion had no effect on tissue MDA content or CuZn-SOD, GDP and GRD activity, and in general, PEG-SOD also lacked significant effect on any of these variables at any dose studied. However, Mn-SOD activity was severely reduced by ischaemia and reperfusion (from 42 +/- 7 U/mg protein in fresh aerobic controls to 6 +/- 1 U/mg protein at the end of reperfusion).(ABSTRACT TRUNCATED AT 400 WORDS)


Iron-catalyzed reactions cause lipid peroxidation in the intact heart.

The chemical targets and mechanisms of iron-catalyzed oxidative injury in myocardium are poorly understood. Oxygen metabolites, in the presence of iron, can initiate free-radical chain reactions in unsaturated membrane lipids, generating lipid peroxides and causing membrane injury. We examined whether exposure to iron-catalyzed oxidative injury would increase myocardial lipid peroxide levels as injury evolved in the intact heart. Isolated, buffer perfused rabbit hearts were exposed for 30 min to 100 uM Fe2+/500 uM ADP and 10 uM H2O2 (IRON group, n = 5), saline vehicle (CON group, n = 6) or 500 uM ADP and 10 uM H2O2 without iron (ADP, n = 5). Lipid peroxides were measured in cytosol and membrane fractions by a new method, using the lipid peroxide-induced oxidation of exogenous GSH to GSSG, catalyzed by the enzyme glutathione peroxidase. The results indicated that iron-catalyzed lipid peroxidation occurs in the intact heart during chemically-mediated oxidative injury.


Stimulation of DNA synthesis by PDGF in the newt cardiac myocyte.

The adult newt cardiac ventricular myocyte has been successfully placed in cell culture and has been shown to undergo in vitro DNA synthesis. Although several growth factors have been reported to increase DNA synthesis in cardiac myocytes in vitro, PDGF has not been reported to do so, but has been shown to be active in other systems. Ventricles were removed from the adult red-spotted newt and were enzymatically and mechanically dissociated in a solution containing trypsin and collagenase. Cells were preplated on to plastic to remove non-myocytes. Myocytes were then plated onto laminin. Groups of myocytes were fed control medium and medium containing porcine PDGF. Myocytes were given 1 microCi/ml of tritiated thymidine 6 or 24 h before fixation. Control myocytes showed a peak DNA synthesis at 12-14 days in culture. One ng/ml of PDGF increased DNA synthesis significantly to 22% above control. Myocytes responded to PDGF with significantly increased DNA synthesis in about 12 h. PDGF did not induce earlier DNA synthesis, but increased synthesis at all days of culture tested. These results indicate that PDGF acts upon cardiac myocytes, increasing their DNA synthesis.


Effects of sympathetic innervation on size of myocytes in embryonic rat heart cultured in oculo.

Embryonic rat myocardium cultured in the anterior eye chamber of an adult rat increases in mass and differentiates into mature myocardium by most morphological criteria [1]. When sympathetic innervation of grafted heart tissue was prevented by superior cervical ganglionectomy (SCGx), growth of the grafts was severely compromised. The present study used morphometric methods to examine the contribution of myocyte size to the differential growth of grafts in sympathetically denervated and intact eye chambers. For this purpose, atria or ventricles from 12-day gestation rat hearts were grafted into sympathetically denervated and intact eye chambers of male host rats. Tissue was harvested after either 2 or 8 weeks in oculo. Myocyte diameter and the ratio of cytoplasmic area to nuclear area increased between 2 and 8 weeks in oculo in both atrial and ventricular grafts. In these unloaded heart grafts, estimated myocyte size did not differ between atrial and ventricular myocytes. Grafts into sympathetically innervated and denervated eye chambers did not differ in myocyte diameter or in the ratio of cytoplasmic to nuclear area, suggesting that myocyte size cannot explain the smaller mass of grafts in sympathetically denervated eye chambers. Thus, it is likely that other factors such as myocyte proliferation, myocyte survival or altered tissue composition underlie the lesser growth of grafts not innervated by sympathetic nerves.


Positive and negative inotropic effects of phorbol 12-myristate 13-acetate: relationship to PKC-dependence and changes in [Ca2+]i.

The present study examined the concentration-dependent effects of phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, on contractile force and [Ca2+]i in guinea-pig hearts and isolated cardiac myocytes, respectively. Contractile force was measured using isolated Langendorff-perfused hearts while [Ca2+]i was measured independently in isolated cardiac myocytes loaded with fura2-AM. Phorbol 12-myristate 13-acetate, as well as another PKC-activating phorbol, phorbol dibutyrate (PDBu), and two non-PKC-activating phorbols, alpha-phorbol didecanoate (alpha PDD) and 4 alpha-phorbol, exerted time- and concentration-dependent effects on contractility. A significant positive inotropic response was observed with either PMA (10(-12) M; 5-15 min of perfusion) or PDBu (10(-12) M; 5 min of perfusion). In contrast, 10(-10) M PMA caused a significant negative inotropic effect following 30 min of perfusion while 10(-8) M PMA produced a significant negative inotropic effect which occurred earlier (10 min) and was sustained throughout the 30 min perfusion period. A similar negative inotropic effect was seen with 10(-8) M of either PDBu or alpha PDD. In addition, 4 alpha-phorbol (10(-8) M) exerted a modest, but significant negative inotropic effect following 25 and 30 min of perfusion. Both concentration-dependent increases and decreases of +dF/dt and -dF/dt were observed in the presence of PMA. In addition, both PMA and PDBu caused a concentration-dependent increase in coronary perfusion pressure. The positive inotropic responses and coronary perfusion pressure effects elicited by PMA and PDBu were largely prevented by the addition of the PKC inhibitors H7 (6 nM) or HAG (10 nM); however, these drugs were without effect on the negative inotropic response to higher concentrations of both PKC-activating (PMA, PDBu) and non-PKC-activating (alpha PDD, 4 alpha-phorbol) phorbol compounds. The lowest concentration of either PMA or PDBu (10(-12) M) increased the 340/380 fluorescence ratio of isolated cardiac myocytes loaded with fura2-AM on a time scale similar to that at which the positive inotropic response was seen in the whole heart. However, in contrast to results in the isolated heart, PDBu elicited a greater and sustained increase in the fluorescence ratio measured in isolated cardiac myocytes. The higher concentration of either PMA or PDBu (10(-8) M), resulted in a decrease in the 340/380 ratio.(ABSTRACT TRUNCATED AT 400 WORDS)


Human fetal heart development after mid-term: morphometry and ultrastructural study.

A total of 44 normally developed human fetal hearts aged from 17 to 40 weeks gestation were provided for routine ultrastructural and morphometric studies. For morphometric analysis, the maximal thicknesses of the anterior, lateral and posterior wall of both ventricles and that of interventricular septum were measured and the left-to-right thickness ratios calculated. The cross-sectional areas of both ventricles in the plane of the greatest heart diameter were measured with computerized image analysis system. Data were analyzed using linear regression and one-way analysis of variance. Myofibril formation occurred by attachment of thin filaments into amorphous Z materials which were presented in sarcolemmal plaques, sarcoplasmic condensations, desmosomes and in Z lines. From these Z centers, myofibrils radiated many directions and branched and anastomosed with further development. This pattern of myofibrillar development continued throughout the whole fetal period. A transverse tubule system was clearly evident in later fetal development. It occurred by invagination of sarcolemma into myocardial cells and formation of subsarcolemmal caveolae. Mitochondria, well-developed Golgi complexes, glycogen granules and well-developed microvessels were found throughout the whole fetal period. Binucleated myocytes appeared by 32 weeks gestation and this suggests that myocyte hyperplasia may cease before birth in humans. The growth of both ventricular walls, the interventricular septum and that of both ventricular cross-sectional areas showed linear regression, and the left-to-right wall thickness ratios were nearly constant. Also, there were no differences in morphometric data between the left and right ventricles. In conclusion, development of the myocyte is an ongoing process which may be continued in the post-natal period in humans, and our statistical results do not support the theory of the right ventricular dominance during the fetal period.


Lysophosphatidylcholine-induced Ca(2+)-overload in isolated cardiomyocytes and effect of cytoprotective drugs.

It has been previously demonstrated that lysophosphatides accumulate rapidly in ischaemic tissue, and may play a key role in the genesis of ischaemia-reperfusion injury. The present study investigated the effects of exogenously added lysophosphatidylcholine (1-20 microM) on single isolated cardiomyocytes from adult rabbit hearts. Quiescent cells exposed to > or = 8 microM lysophosphatidylcholine dose-dependently displayed irreversible hypercontraction, whereas after 60 min at 3 microM lysophosphatidylcholine, most cells remained rod-shaped (87.2 +/- 2.0%, mean +/- S.E.M.). However, when combined with electrical field stimulation (1 Hz), exposure to 3 microM lysophosphatidylcholine resulted in irreversible hypercontracture of most cells after 60 min: only 27.5 +/- 7.5% of the cells remained rod-shaped. Contracture depended upon the presence of extracellular Ca2+, and coincided with a significant rise in the median intracellular free Ca2+ level from 72.2 to 352.1 nM (P = 0.0001), suggesting intracellular Ca(2+)-overload. Pretreatment with 10(-6) M flunarizine or R 56865 significantly reduced the fraction of damaged cells when exposed to 3 microM lysophosphatidylcholine and electrical stimulation: 78.3 +/- 12.2% and 56.3 +/- 13.1% respectively of the cells remained rod-shaped. No protection was observed when quiescent cells were exposed to 10 microM lysophosphatidylcholine. Cytochemical localization of Ca2+ showed that lysophosphatidylcholine induced a loss of sarcolemma-bound Ca2+ precipitate and an accumulation of Ca2+ clusters in mitochondria of damaged cells in a dose and time dependent way. These results suggest that lysophosphatidylcholine induces functional and structural damage (Ca(2+)-overload) in isolated cardiomyocytes and that this can be prevented by cytoprotective drugs.


Cardiomyocytes express albumin binding proteins.

We investigated whether cardiomyocytes express specific albumin binding proteins (ABP) which may function in the dissociation of fatty acids from their non-covalent complexes with albumin. The experiments were performed on rat neonatal cardiomyocytes (freshly isolated and up to 3 days in culture) and on an enriched sarcolemmal fraction isolated from adult rabbit ventricular myocardium. Three types of experiments were conducted: (a) identification of ABP on electroblots of cardiomyocytes and sarcolemmal extracts reacted with [125I]-bovine serum albumin ([125I]Alb); (b) kinetic assays of [125I]Alb interaction with cardiomyocytes (at 37 degrees C), and with a sarcolemmal fraction (at 4 degrees C); (c) affinity isolation of ABP from solubilized radioiodinated sarcolemmal proteins interacted with an albumin-agarose matrix. The investigation showed that: first, two pairs of polypeptides (ABP of M(r) 18 and 31 kDa) in either cardiomyocytes or sarcolemmal fraction reacted on electroblots with [125I]Alb; second, the binding of the latter to cardiomyocytes was saturable and competed by unlabeled albumin: 50 microM albumin reduced by approximately 90% the binding of radiolabeled albumin. The sarcolemmal fraction bound [125I]Alb with a Kd of 3.66 x 10(-7) M. Thirdly, among the sarcolemmal proteins retained by the albumin-agarose matrix (18 and 31 kDa), the most prominent was the lower band (approximately 16 kDa) of the 18 kDa pair of ABP. The observations revealed that albumin interacts with relatively high affinity with specific binding sites on cardiomyocyte sarcolemma. This interaction may be a recognition step for subsequent fatty acid dissociation and translocation.


Risk factors for fatal colon cancer in a large prospective study.

Diet, physical activity, obesity, aspirin use, and family history may all modify the risk of colon cancer, but few epidemiologic studies are large enough to examine these factors simultaneously. We prospectively assessed the relationship of diet and other factors to risk of fatal colon cancer. Using data from Cancer Prevention Study II--an ongoing prospective mortality study--we studied 764,343 adults who, in 1982, completed a questionnaire on diet and other risk factors and did not report cancer or other major illness. We assessed mortality through August 1988 and identified 1150 deaths from colon cancer (611 men and 539 women). Multivariate analyses were used to compare these case patients with 5746 matched control subjects drawn from the cohort. Risk of fatal colon cancer decreased with more frequent consumption of vegetables and high-fiber grains (P for trend = .031 in men and .0012 in women). The relative risk (RR) for the highest versus lowest quintile of vegetable intake was 0.76 in men (95% confidence interval [CI] = 0.57-1.02) and 0.62 in women (95% CI = 0.45-0.86). Dietary consumption of vegetables and grains and regular use of aspirin were the only factors having an independent and statistically significant association with fatal colon cancer. Participants who consumed the least vegetables and grains and no aspirin had a higher risk compared with those who consumed the most vegetables and used aspirin 16 or more times per month. For men in the former category, the RR was 2.4 (95% CI = 1.1-5.3); for women, it was 2.9 (95% CI = 1.3-6.7). Weaker associations were seen for physical inactivity, obesity, total dietary fat, and family history. No associations were seen with consumption of red meat or total or saturated fat in either sex, but this finding must be interpreted cautiously. These findings support recommendations that increased consumption of vegetables and grains may reduce the risk of fatal colon cancer. Regular use of low doses of aspirin may prove to be an important supplemental measure.


Cancer incidence and survival among American Indians registered for Indian health service care in Montana, 1982-1987.

Cancer incidence and cancer survival estimates in American Indians are quite limited. Our purpose was to estimate cancer incidence and survival in American Indians who were registered for Indian Health Service (IHS) care in Montana. We linked databases from the IHS and the Montana Central Tumor Registry (MCTR) to ascertain cases for the time period from January 1, 1982, through December 31, 1987. To calculate survival rates, we used a relative survival method that incorporated age-specific risks for noncancer deaths among American Indians. We identified 344 cases that were compatible with the National Cancer Institute (Surveillance, Epidemiology, and End Results Program) surveillance definition of cancer. Of these cases, 249 (72%) were listed in both the MCTR and the IHS databases; 56 (16%) and 39 (11%) were listed in only the MCTR or the IHS database, respectively. Compared with the overall cancer incidence in U.S. White men, the overall cancer incidence in Montana American Indian men was markedly lower, as was the incidence for colorectal and bladder cancers and for non-Hodgkin's lymphoma. The overall cancer incidence for Montana American Indian women differed less markedly, however, from the overall incidence in U.S. White women. Compared with the cancer incidence in U.S. White women, the incidence in Montana American Indian women was significantly higher for cervical cancer but was significantly lower for colorectal, breast, and uterine cancers. Survival rates from cancer were also examined for the first time in this population. For those sites examined, the survival rates were much lower in Montana American Indians than in U.S. Whites. We conclude that it is feasible to develop state-specific cancer incidence and survival estimates for American Indians in at least some states in different regions of the United States. Collaboration between the IHS and a state tumor registry is likely to improve the case ascertainment achieved by either agency alone.


ERCC1 and ERCC2 expression in malignant tissues from ovarian cancer patients.

ERCC1 and ERCC2 are human DNA repair genes that are associated with in vitro resistance to selected DNA-damaging agents. Fresh tumor tissues from 26 patients with ovarian cancer were analyzed for the RNA levels of expression of these genes to determine possible clinical relevance. Tumor tissues were harvested from patients immediately before they entered a cisplatin- or carboplatin-based treatment protocol. Clinical response was assessed by standard criteria. Gene expression level was assessed by slot blot analysis, using beta-actin as a control. Relative expression levels were determined by comparing each tumor sample with a Chinese hamster ovary cell line that had a stable transfection of the human ERCC1 gene. Patients who were clinically resistant to platinum-based therapy had a 2.6-fold higher expression level of ERCC1 in their tumor tissue than did patients who responded to that therapy (P = .015). Results obtained by slot blot analysis were qualitatively confirmed by polymerase chain reaction analysis. Relative levels of expression of ERCC2 did not differ significantly between responders and nonresponders. We conclude that ERCC1 expression levels in human tumor tissue may have a role in clinical resistance to platinum compounds. These data appear to be consistent with the assertion that ERCC1 serves as an excision nuclease, whereas ERCC2 serves as a helicase.


Comparative sensitivity of cells from human tumors and derivative tumor xenografts to radiation and heat treatments.

Human tumor xenografts in athymic mice are often used to study the efficacy of cancer treatments, but it is not known whether the xenografts accurately reflect the treatment responsiveness of the original tumors. To assess the validity of tumor xenografts as specific models of neoplastic disease, a soft-agar colony assay was used to compare the sensitivity of cells from surgical tumor specimens and derivative xenograft lines to various radiation and heat treatments in vitro. Xenograft lines were established from melanomas and from carcinomas of the bladder, breast, cervix, and colon. Single-cell suspensions from the original tumors and the corresponding tumor xenografts were subjected to treatments that measured inherent radiation sensitivity, split-dose radiation repair capacity, low-dose-rate radiation sensitivity, inherent heat sensitivity, capacity for development of thermotolerance, and sensitivity to step-down heating (treatment at 43.5 degrees C followed by treatment at 41.5 degrees C). Cell survival curves were plotted for each treatment, and a two-tailed Student's t test was used to search for statistically significant correlations between the curves. Although cells from different surgical tumor specimens varied widely in their sensitivity to radiation and heat treatments, there was always a statistically significant correlation (P < .05) between the sensitivity of cells from the surgical specimens and the corresponding tumor xenografts. These observations strongly suggest that intrinsic properties of human tumor cells generally are retained during serial heterotransplantation. Thus, human tumor xenograft lines may be valuable tools in studies aimed at identifying cellular properties of importance for the clinical treatment sensitivity of human cancer. This does not necessarily mean, however, that the treatment responsiveness of the xenografts in vivo mirrors the clinical treatment responsiveness of the tumors in patients.


ras oncogene activation and occupational exposures in acute myeloid leukemia.

Epidemiologic studies of acute myeloid leukemias (AMLs) show small increases in risk of disease associated with certain occupations and chemical exposures. This study was designed to determine whether the presence of mutationally activated ras oncogenes in AML are associated with occupational and chemical exposures. We interviewed 62 patients with newly diagnosed AML (or their next-of-kin), all of whom were enrolled in a national multicenter clinical trial, and 630 healthy control subjects. DNA extracted from patients' pretreatment bone marrow samples was amplified by using the polymerase chain reaction and probed with allele-specific oligonucleotides for activating point mutations at the 12th, 13th, and 61st codons of three protooncogenes: H-ras (also known as HRAS), K-ras (also known as KRAS2), and N-ras (also known as NRAS). Patients with ras mutation-positive AML had a higher frequency (six of 10 patients) of working 5 or more years in an a priori high-risk occupation than did patients with ras mutation-negative AML (eight of 52; odds ratio [OR] = 6.8; 95% confidence interval [CI] = 1.3-36). Patients with ras mutation-positive AML were more likely than patients with ras mutation-negative AML to have breathed chemical vapor on the job (OR = 9.1; 95% CI = 1.3-64) or to have had skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37). When ras-positive patients were compared with healthy control subjects, the ORs for occupation and occupational exposures remained elevated, while patients with ras mutation-negative AML showed no increased risk when compared with control subjects. Activation of ras proto-oncogenes may identify an etiologic subgroup of AML caused by occupation and chemical exposure. Disease etiology may be better understood if epidemiologic measures of exposure are integrated with molecular assays of the genetic defects responsible for cancer initiation and promotion.


Simultaneous high-resolution measurement of adenosine triphosphate levels and blood flow in the hamster amelanotic melanoma A-Mel-3.

Some studies suggest that nutritional blood flow and intratumor levels of high-energy phosphates such as adenosine triphosphate (ATP) affect tumor response to treatment. Reports of intratumor variabilities have shown that distribution of high-energy phosphates within experimental and human tumors is not uniform, and this variability may be related to differences in blood flow in different regions of the tumor. However, previous studies provide no insight into the intratumor relationship of these parameters. To study the intratumor relationship between blood flow and ATP concentration, we have developed a method that allows simultaneous quantitation of the two variables almost at a cellular level at adjacent sites. In addition, this technique, which uses digital image processing and analysis, facilitates regional high-resolution measurements of blood flow and ATP concentrations in relation to histology. We examined tissue samples cut in consecutive order from one representative hamster A-Mel-3 amelanotic melanoma. Specimens from the same tumor were analyzed for histological features, blood flow (measured autoradiographically by the use of [4-N-methyl-14C]iodoantipyrine), and ATP content (measured by a quantitative substrate-induced bioluminescence reaction, using single-photon imaging). The data on the three parameters were stored as digitized images, which were later transformed to reach geometric congruency. The relationship between blood flow and ATP was examined by nonlinear regression and correlation analyses. Regional distribution of blood flow was highly correlated with distribution of ATP within the A-Mel-3 tumor. For images of the tumor using measuring fields of approximately 20 tumor cells, the coefficient of correlation was .92 (P < .001). The relationship between the two parameters was described by a second-order function. At low blood flow values, small differences in blood flow were associated with large changes in levels of ATP, whereas at high blood flow values, differences in blood flow were associated with much smaller changes in ATP levels. This high-resolution technique may have applications in future studies investigating the relationship between tumor cell metabolism and regional blood flow and in studies aimed at identifying the locoregional target of various cancer therapies.


Current serum levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin in phenoxy acid herbicide applicators and characterization of historical levels.

Workers who sprayed phenoxy acid herbicides, especially those who sprayed before 1975, may have been exposed to significant amounts of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent animal carcinogen present in herbicide preparations as a contaminant. The aims of this study were (a) to determine serum levels of TCDD in a representative sample of workers occupationally exposed to the agent during the spraying of phenoxy acid herbicides; (b) to compare serum levels in workers exposed before 1965, when concentrations in herbicide products were unregulated and high, with levels in workers exposed after 1974, when concentrations were lower as a result of government regulations worldwide; and (c) to examine the correlation, if any, between serum levels and duration of employment in spraying. Thirty-seven subjects were randomly selected from a group of 654 men who had sprayed the herbicides 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Australia for at least 12 months. The workers were classified as follows: eight who sprayed only before 1965, nine who sprayed only during the period after 1964 and before 1975, and 20 who sprayed during the period after 1974 and before 1991. Serum from the workers was analyzed for TCDD by high-resolution gas chromatography and high-resolution mass spectrometry at a detection limit of 0.6 parts per trillion (ppt) on a lipid-weight basis. In addition, rates of exposure to TCDD were estimated, as were TCDD serum concentrations at termination of employment and intensity of herbicide use. Only one worker, with a serum TCDD level of 34 ppt, had a serum level higher than the maximum level of 26 ppt reported for the general population. Assuming a half-life of 7.1 years, we estimated the mean exposure rates to be 2.7, 2.3, and 0.06 ppt/mo for the three epochs, respectively. We found the highest serum level of TCDD at the time of cessation of employment to be 329 ppt. Calendar period and intensity of use of 2,4,5-T and 2,4-D were statistically significant determinants of rate of exposure to TCDD, but 2,4-D was associated with exposure rate only for the pre-1975 periods. Estimated rates prior to 1965 were more than an order of magnitude higher than those after 1974. The highest estimated exposure rate was 20.7 ppt/mo, which suggests that some sprayers may have been exposed to levels comparable with those that produce cancer in laboratory animals.


Effects of verapamil on the acute toxicity of doxorubicin in vivo.

Studies indicating that verapamil substantially enhances doxorubicin levels in certain drug-resistant tumor cells have led to the use of verapamil in combination with doxorubicin in animal and clinical studies of multidrug-resistant tumors. These studies have shown this drug combination to be associated with severe toxic effects. It is important to determine whether verapamil modulates the dose-limiting and potentially lethal cardiotoxicity of doxorubicin and to elucidate possible mechanisms. The aims of this study were to evaluate the in vivo effects of verapamil on (a) doxorubicin-stimulated cardiac lipid peroxidation and cardiac damage, (b) doxorubicin-induced animal mortality, and (c) biodistribution of doxorubicin to the heart. Male (BALB/c x DBA/2)F1 mice were treated with a high dose of doxorubicin (15 mg/kg, injected intraperitoneally), verapamil (25 mg/kg, injected intraperitoneally), or combinations of the two. Lipid peroxidation was determined using the 2-thiobarbituric acid assay for malonaldehyde. Light microscopy was used for histopathologic examination of cardiac tissue. A fluorometric assay procedure was employed to determine doxorubicin levels in the heart. Verapamil was an effective inhibitor of peroxidative damage to myocardial lipids following a high dose of doxorubicin (15 mg/kg, injected intraperitoneally). However, mice treated with verapamil and doxorubicin had a lower survival rate and a higher initial peak concentration of doxorubicin in the heart than those treated with doxorubicin alone. They also demonstrated a higher incidence and severity of degenerative changes in cardiac tissue. Our findings suggest that verapamil effectively inhibits doxorubicin-mediated lipid peroxidation in vivo but that cardiac lipid peroxidation is not the major limiting mechanism underlying doxorubicin-induced toxicity. A possible explanation for the excess mortality and cardiac injury in mice treated with verapamil plus doxorubicin is that verapamil alters the pharmacokinetics of doxorubicin. Further studies are necessary for development of safer protocols and/or drug combinations to treat multidrug-resistant tumors. We are currently studying treatment of tumor-bearing animals with a cumulative dosage regimen of doxorubicin in the presence and absence of verapamil.


Pharmacokinetics of the cardioprotector ADR-529 (ICRF-187) in escalating doses combined with fixed-dose doxorubicin.

Although doxorubicin is an anticancer agent with a wide spectrum of activity, therapy with this anthracycline must often be discontinued at a time of benefit to the patient because of the drug's cumulative cardiotoxicity. ICRF-187 (ADR-529, dexrazoxane) is a bisdioxopiperazine compound that protects against cardiac toxicity induced by doxorubicin. Our objectives in this study were to determine the maximum tolerated dose of ADR-529 (which uses a different vehicle than ICRF-187) when given with a fixed doxorubicin dose and to determine whether ADR-529 alters doxorubicin pharmacokinetics. Twenty-five patients were treated with doxorubicin (60 mg/m2) preceded by administration of ADR-529 in escalating dosages (i.e., 60, 300, 600, 750, and 900 mg/m2) to groups of three to nine patients. ADR-529 was administered over a 15-minute period beginning 30 minutes before doxorubicin treatment; the protocol was repeated every 3 weeks. Blood was sampled frequently for drug levels, which were determined by high-pressure liquid chromatography with fluorescence (doxorubicin) and electrochemical detection (ADR-529). Dose-limiting neutropenia occurred in four of six previously treated patients at an ADR-529 dose of 600 mg/m2; the dose ratio of ADR-529 to doxorubicin was 10:1. For three additional patients with better Eastern Cooperative Oncology Group performance status and a maximum of one prior chemotherapy regimen, 600 mg/m2 was tolerated, but grade 3 or 4 neutropenia occurred in four of six patients who received an ADR-529 dose of 900 mg/m2 and in three of four patients at a dose of 750 mg/m2. Doxorubicin's estimated terminal half-life was 39.5 +/- 18.3 (mean +/- SD) hours; the area under the curve for plasma concentration of drug x time (AUC) was 1.74 +/- 0.40 (micrograms/microL) x hour. Total-body clearance was 598 +/- 142 microL/m2 per minute (N = 20), and it did not vary with ADR-529 dose. Estimated distribution and elimination phase half-lives for plasma ADR-529 were 0.46 +/- 0.30 hours and 4.16 +/- 2.94 hours, respectively. Total-body clearance was 111 +/- 87 microL/m2 per minute (N = 18); AUC was linear (r2 = .92), and the clearance rate was constant (r2 = .18) from 60 to 900 mg/m2. Myelotoxicity was dose limiting for ADR-529 at 600-750 mg/m2 when given with a fixed dose of doxorubicin at 60 mg/m2 (dose ratios of ADR-529 to doxorubicin ranged from 10:1 to 12.5:1). When used in combination, ADR-529 did not perturb doxorubicin's distribution, metabolism, or excretion; therefore, other mechanisms of cardioprotection must be involved. We recommend that an ADR-529 dose of 600 mg/m2 be given with single-agent doxorubicin at a dose of 60 mg/m2 in future studies.


Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors.

There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animal in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%, and 73%, respectively; these percentages were not significantly different. Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU-induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis.


Individual variation in the activation and inactivation of metabolic pathways of cyclophosphamide.

Carboxyphosphamide is an inactive metabolite of cyclophosphamide, which is a widely used antineoplastic drug. Deficiencies in the production of this metabolite have been reported. Such deficiencies would have important consequences for therapeutic and toxic effects of oxazaphosphorines like cyclophosphamide. This study further investigates the variability in cyclophosphamide metabolism and carboxyphosphamide recovery in urine. The 24-hour urinary metabolic profile of cyclophosphamide was investigated in 17 Turkish patients receiving doses of 100-1080 mg orally or by short intravenous infusion. Urine samples were assayed quantitatively for cyclophosphamide and its principal metabolites (phosphoramide mustard, 4-ketocyclophosphamide, carboxyphosphamide, and dechloroethylcyclophosphamide) with combined thin-layer chromatography-photography-densitometry. The amount of each metabolite excreted in 24 hours was expressed as a percentage of the dose. Recovery of drug and metabolites varied greatly among individuals (range, 0.01%-13.56% of dose). In particular, the amount of carboxyphosphamide varied over a thousandfold range and was undetectable in urine from four patients. The patients were classified by phenotype as demonstrating low or high carboxylation. Those with low carboxylation excreted less than 0.2% of the cyclophosphamide dose as carboxyphosphamide, while those with high carboxylation excreted 0.8%-13.6% (median, 1.81%). No association was observed between carboxylation phenotype and patient age, sex, disease, or concomitant therapy, although the three lifetime nonsmokers all showed poor carboxylation. No correlation was observed between the percent of dose excreted as any of the other metabolites and that excreted as carboxyphosphamide. There was a statistically significant inverse correlation between the combined recovery of carboxyphosphamide and phosphoramide mustard and the dose of prednisolone administered. These data confirm an earlier observation of a phenotypic deficiency of carboxyphosphamide excretion in British patients treated with cyclophosphamide. This deficiency may arise from a polymorphism in the enzyme aldehyde dehydrogenase. Carboxylation phenotype may have important implications for both the therapeutic effect and toxicity of cyclophosphamide.


Chromosomal abnormalities in patients with Hodgkin's disease: evidence for frequent involvement of the 14q chromosomal region but infrequent bcl-2 gene rearrangement in Reed-Sternberg cells.

Rearrangements of the bcl-2 gene (also known as BCL2) have been detected in up to 40% of cases of Hodgkin's disease, and it has been speculated that such rearrangements may have a role in the pathogenesis of Hodgkin's disease. The purposes of this study were (a) to assess the frequency of clonal chromosomal abnormalities in Hodgkin's disease, (b) to identify recurrent changes, (c) to determine whether the bcl-2 gene rearrangement was present in Reed-Sternberg cells (the neoplastic cells of Hodgkin's disease) and their variants, and (d) to analyze whether the presence of t(14;18) translocations in Reed-Sternberg cells explains the observed bcl-2 gene rearrangements in Hodgkin's disease. A cytogenetic study was performed on biopsy specimens from 28 consecutive untreated patients with Hodgkin's disease. The same patients were analyzed for bcl-2 gene rearrangement by a polymerase chain reaction (PCR) technique. To ascertain whether the abnormal karyotypes were present in and restricted to Reed-Sternberg cells, we also performed in situ hybridization with chromosome-specific probes. Abnormal metaphases were identified in 23 of the 28 patients. In 11 patients, the chromosome 14q region was abnormal; in six of these patients, there was involvement of the 14q32 region that comprises the gene encoding for heavy-chain immunoglobulin. Only one patient had a t(14;18) translocation, whereas almost 40% of these 28 patients showed bcl-2 gene rearrangements by a PCR method. The in situ hybridization method showed that the abnormal karyotype was present in and restricted to Reed-Sternberg cells. We conclude that the majority of cases of Hodgkin's disease contain a clonal population with an abnormal karyotype, comprising the Reed-Sternberg cells. The q32 region of chromosome 14 is frequently involved, but a t(14;18) translocation is extremely infrequent. The occurrence of a bcl-2 gene rearrangement in Hodgkin's disease most likely results from the presence of sporadic, small bystander B lymphocytes that carry the translocation and that also can be frequently detected in reactive lymphoid tissue such as tonsils. Also, a range of different chromosomal translocations may provide growth or survival advantages to Reed-Sternberg cells.


Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation.

It has been suggested that multiple sites of epithelial ovarian carcinoma on the peritoneal surface reflect polyclonal disease arising from multiple primary tumors in the peritoneal mesothelium, rather than monoclonal disease spread by metastases from one primary ovarian cancer. The purpose of this study was to investigate whether ovarian cancer has a monoclonal or polyclonal origin. DNA specimens were obtained from peripheral blood lymphocytes (normal DNA) and from multiple tumor deposits of 17 women with epithelial ovarian carcinoma: primary tumors, metastatic deposits, and ascites. The clonal origin of each tumor was determined by performing (a) analysis to detect loss of heterozygosity at five loci on chromosomes 5, 11, 13, and 17; (b) sequencing of exons 5-8 of the p53 gene; and (c) X-chromosome inactivation analysis of the phosphoglycerate kinase (PGK) gene. In 15 of the 17 cases analyzed, there was clear evidence of monoclonal origin. The probability that the genetic events documented in these 15 cases occurred as independent events in each tumor deposit ranged from 2.5 x 10(-1) to 3.7 x 10(-16). In two cases, the pattern of allelic deletion and p53 gene mutation was compatible with either a monoclonal origin or origin from two primary ovarian tumors. The results did not support the hypothesis that ovarian cancer is a multifocal, polyclonal disease. Instead, the data suggest that sporadic epithelial ovarian carcinoma has either a monoclonal or a dual primary origin. These findings have important implications for understanding of the natural history of ovarian cancer and for clinical strategies aimed at prevention and early detection. Further studies will be required to determine the clonal origin of familial hereditary ovarian cancer.


Antitumor activity and clinical pharmacology of sulofenur in ovarian cancer.

Sulofenur is a diarylsulfonylurea with demonstrated antitumor activity in patients with advanced epithelial ovarian cancer refractory to standard chemotherapy. The dose-limiting toxic effects observed in phase I clinical trials have been anemia and methemoglobinemia, resulting in cyanosis. The purposes of this study were to further define the response rate, toxic effects, and pharmacokinetics and pharmacodynamics of sulofenur in patients with advanced ovarian cancer. We conducted a phase II trial of sulofenur at a dose of 800 mg/m2 per day in 35 patients with stage III or IV ovarian cancer refractory to standard chemotherapy. Pharmacokinetics and pharmacodynamics were analyzed by comparing sulofenur parent and metabolite plasma levels with methemoglobin levels. Partial responses lasting 6.5-18 weeks occurred in four (15%; 95% confidence interval = 4%-35%) of the 26 patients assessable for response. In addition, 42% (11) of the assessable patients had prolonged stable disease (median, 20 weeks). The first nine patients received sulofenur as a daily oral dose for 14 days, with a 21-day treatment cycle. However, they developed substantial anemia and methemoglobinemia. As a result, the next 26 patients received sulofenur daily for 5 days followed by 2 days of rest for 3 consecutive weeks, with a 28-day treatment cycle (5/2-day schedule). Preclinical models predicted that 2 days of rest would decrease toxicity while maintaining antitumor activity. Patients treated with the 5/2-day schedule had relatively less severe anemia and methemoglobinemia and needed fewer red blood cell transfusions (31% versus 78% of patients), but 31% still required dose reductions because of these toxic effects. The hydroxy and keto metabolites of sulofenur had prolonged plasma half-lives relative to the parent compound, and the difference was statistically significant. In addition, the correlations of metabolite concentrations with methemoglobin levels were higher than the correlation of sulofenur concentrations with methemoglobin levels, and those differences were statistically significant. We conclude that sulofenur has modest clinical activity in heavily pretreated patients with ovarian cancer. The toxic effects of anemia and methemoglobinemia may limit the ultimate clinical utility of diarylsulfonylureas until less toxic derivatives with alternate metabolic pathways can be identified.


Plasminogen activator and its inhibitor in cancer patients treated with tumor necrosis factor.

We noted the presence of plasma fibrin degradation products in patients treated with recombinant human tumor necrosis factor (TNF) in a phase I trial. To further define this observation, we investigated the effects of TNF on the fibrinolytic system in patients entered in the same trial. In the 14 patients studied, fibrinolytic parameters were measured by analyzing blood samples for tissue plasminogen activator and inhibitor at 0, 1, 2, 4, 6, and 18-24 hours after initiation of TNF treatment. We used a chromogenic substrate method to determine activity of plasminogen activator and its inhibitor and an enzyme-linked immunosorbent assay (ELISA) to determine levels of antigen (tissue-type plasminogen activator). Molecular weight was determined by zymographic assay. TNF treatment was associated with tissue-type plasminogen activator induction within 1 hour of TNF initiation. The plasminogen activator produced was consistent with tissue-type plasminogen activator derived from endothelium as evidenced by molecular weight analysis and ELISA. Moreover, induction of plasminogen activator inhibitor occurred following the release of tissue-type plasminogen activator, and our data suggest a dose-response effect for TNF. At high doses (i.e., 200 and 240 micrograms/m2), there was a more rapid and prolonged release of plasminogen activator inhibitor, which had an inverse relationship with the level of antigenic tissue-type plasminogen activator. Zymographic analysis showed urokinase-type plasminogen activator activity in 13 of 14 patients. In three patients, simultaneous measurements of white blood cells and tissue-type plasminogen activator revealed a temporal association between the TNF-associated rapid granulocytopenia at 30 minutes after TNF initiation and release of tissue-type plasminogen activator antigen. The results suggest a positive association between TNF and rapid induction of plasminogen activator activity that is consistent with an endothelial product. It is possible that, at high doses, TNF may interact directly with vascular endothelium, leading to rapid and prolonged production of plasminogen activator inhibitor. There was a dose-response effect between TNF and release of tissue-type plasminogen activator. The release of tissue-type plasminogen activator was preceded by granulocytopenia, which may indicate an association between a proposed TNF-induced granulocyte-endothelial interaction in vivo and release of tissue-type plasminogen activator. These findings demonstrating the effects of TNF on the fibrinolytic system can be analyzed further in experimental systems to determine the implications for use of this agent as a biological response modifier in cancer therapy.


Decreased catabolism of fluorouracil in peripheral blood mononuclear cells during combination therapy with fluorouracil, leucovorin, and interferon alpha-2a.

We previously reported that recombinant interferon alpha-2a (IFN alpha-2a) therapy was associated with a dose-dependent decrease in fluorouracil (5-FU) clearance. In this study, we used peripheral blood mononuclear cells (PBMCs), which are responsive to IFNs, as surrogate tissue to determine whether the change in clearance might be explained by decrease in 5-FU catabolism during IFN alpha-2a therapy. The study population consisted of 45 patients with adenocarcinoma arising in the gastrointestinal tract. Thirty-seven patients received therapy containing IFN alpha-2a at a median dose of 5 million U/m2 per day (range, 1.7-7.5 million U/m2 per day) starting on day 1 and continuing through either day 7 or day 14 in conjunction with intravenous high-dose leucovorin (LV) followed by bolus 5-FU on days 2-6. Eight patients received the same schedule of 5-FU and LV daily for 5 days without IFN alpha-2a but with granulocyte-macrophage colony-stimulating factor starting on day 6 and ending at least 3 days prior to the start of the next cycle. Peripheral blood was collected during 70 cycles on days 1, 2, and 4 prior to the daily treatment with IFN alpha-2a + 5-FU+LV and during 19 cycles on days 1 and 4 prior to the daily treatment with 5-FU+LV without IFN alpha-2a. In a given patient cycle, matched samples were drawn at approximately the same time of day. PBMCs were isolated, and the intact cells were exposed to 4 microM [3H]5-FU, and the formation of [3H]dihydrofluorouracil was determined by reverse-phase high-performance liquid chromatography. In 47 matched patient cycles from IFN alpha-2a + 5-FU+LV-treated patients in which samples were available on days 1, 2, and 4, 5-FU catabolism decreased by 20% (P2 = .03) and 41% (P2 = .0001) from the baseline catabolic rate (2.5 +/- 0.2 pmol/min per 10(6) cells [mean +/- SE]) on days 2 and 4, respectively. Using information from all paired samples, the mean change from baseline on day 2 was -0.4 +/- 0.2 pmol/min per 10(6) cells (n = 54; P2 = .05), and the change from baseline on day 4 was -1.3 +/- 0.3 pmol/min per 10(6) cells (n = 63; P2 = .0001). In contrast, changes in 5-FU catabolism were not evident in the PBMCs of the reference population receiving 5-FU+LV without IFN alpha-2a. The magnitude of the change in 5-FU catabolism is similar to the magnitude of the decrease in 5-FU clearance in our previous study. These observations suggest that changes in 5-FU catabolism during therapy with IFN alpha-2a, 5-FU, and LV may account for the decreased 5-FU clearance.


Can thymidine kinase levels in breast tumors predict disease recurrence?

Our previous study of thymidine kinase (TK) levels in the serum of breast cancer patients demonstrated a statistically significant positive correlation with cancer stage. In postsurgical follow-up studies of 20 patients with primary breast cancer, total serum TK levels rose with disease recurrence and continued to rise with disease progression but decreased with treatment response. This study was designed to examine whether TK levels in primary breast tumors can be used to predict recurrence and to establish the relationship between TK levels and estrogen receptor (ER) status and recurrence. Eighty-six patients with breast cancer were entered in this study. Tumors were assessed for ER status and TK levels, and the patients had follow-up for recurrence over a period of 41 months. By calculating the percent of TK activity in the presence of adenosine triphosphate (ATP) or cytidine triphosphate (CTP), we estimated the relative contributions of TK isozymes TK1 and TK2 to total TK activity. Total TK (TK1 plus TK2) levels in tumors were significantly (P < .001) elevated in patients who subsequently had recurrence compared with levels in those who did not. Calculations of the percent of TK activity in the presence of ATP or CTP showed that this elevation was due to increased TK1 isozyme levels. ER-negative (ER-) patients had significantly (P < .001) increased TK1 levels relative to those in ER-positive (ER+) patients. ER- patients with recurrence had significantly (P < .001) elevated total tumor TK levels compared with levels in those who did not have recurrence, and calculation of percent of TK activity with ATP or CTP indicated elevated TK1 levels. A similar pattern of increased levels of total tumor TK and TK1 was observed in ER+ patients with recurrence. The results indicate that total tumor TK levels were significantly higher in breast cancer patients who subsequently had recurrence than levels in those who did not. This finding appears to be largely caused by higher levels of TK1. Higher TK1 levels in tumors in patients who subsequently had disease recurrence almost certainly indicate a high rate of proliferation in such tumors at the time of excision. It appears that TK is a potentially useful marker in the management of breast cancer. With measurement of levels of TK, particularly TK1, in breast tumors and serum, it may be possible to predict recurrence of breast cancer.