13C NMR of cyanylated flavodoxin from Megasphaera elsdenii and of thiocyanate model compounds.

Both of the thiol groups of Megasphaera elsdenii flavodoxin have been cyanylated using 13C-enriched cyanide. This chemical modification increases the dissociation constant of the apoflavodoxin-flavin mononucleotide (FMN) complex from 0.4 nM to 2 microM. The thiocyanate carbons of the cyanylated cysteine residues in apoflavodoxin had 13C chemical shifts of 109.4 ppm and 112.2 ppm, which were replaced by signals at 115.5 ppm and 109.6 ppm when FMN was bound. The signals at 109.4 ppm and 112.2 ppm due to the cyanylated apoflavodoxin were unstable at 28 degrees C, and they were slowly replaced signals at 114.5 ppm and 115.3 ppm which are attributed to an inactive form of the apoprotein, which does not bind FMN. At alkaline pH values or after prolonged incubation at neutral pH, the signals at 114.5 ppm and 115.3 ppm were replaced by signals at approximately 171 ppm. On the basis of results obtained with model compounds, the signals at 171 ppm are assigned to the 2-imino carbon of the 2-iminothiazolidine ring formed by the cyclization of the appropriate thiocyanate group. After determining the chemical shift of the thiocyanate carbon of model compounds in a range of solvents, we conclude that the thiocyanate carbons will have a minimal chemical shift of approximately 109 ppm in apolar solvents which do not contain hydrogen bond donors. In water, a more polar hydrogen-bonding solvent, the chemical shift increases to approximately 115 ppm. We also conclude that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment.(ABSTRACT TRUNCATED AT 250 WORDS)


Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)


Orientation factor in steady-state and time-resolved resonance energy transfer measurements.

Resonance energy transfer measurements provide a way to estimate distances between chromophores attached to different sites of macromolecules. There are two unknowns involved in resonance energy transfer measurements, the distance between two chromophores and their relative orientation. When static orientational disorder exists, the orientation factor, kappa 2, can vary from 0 to 4, leading to considerable uncertainty in estimation of distances. Fluorescence polarization anisotropy measurements can reduce the degree of uncertainty [Dale & Eisinger (1974) Biopolymers 13, 1573]. There may still be substantial error bounds for the average distance measurements. Time-resolved fluorescence measurements provide an "apparent" average distance and distance distribution containing contributions by both distance and orientation. The contribution of orientation to observed "apparent" average distance and distance distribution widths has been estimated for both simulated and real data. With a single unique distance as input in the simulation and with random but static orientation of donor and acceptor, the recovered average distance is very close to that of the input when the input distance is close to or larger than the Förster distance. The recovered width of apparent distance distribution can be substantial and it changes as a function of Förster distance to average distance ratio and as a function of Förster distance. Similar conclusions apply to the case where there is a real distance distribution. Motional averaging of the orientation was simulated by the Monte Carlo method to estimate the contribution of orientation when chromophores have certain degrees of mobility.(ABSTRACT TRUNCATED AT 250 WORDS)


Tryptophan contributions to the unusual circular dichroism of fd bacteriophage.

The circular dichroism (CD) spectrum of fd bacteriophage has a deep minimum at 222 nm characteristic of highly alpha-helical protein, but there is a shoulder at 208 nm rather than a minimum, with a 222/208-nm amplitude ratio near 1.5 rather than near 1. Oxidation of fd phage with the tryptophan reagent N-bromosuccinimide (NBS) changes the ratio. In this report, the NBS titration of fd is followed by CD and three other spectroscopies, the results of which yield an explanation of the unusual CD spectrum. Absorbance, fluorescence, and Raman data show the oxidation to have two phases, the first of which involves the destruction of tryptophan and the second, tryptophan and tyrosine. Raman spectra reveal the invariance of an environmentally-sensitive tyrosine Fermi resonance doublet during the first oxidative phase. Raman spectra also show that little or no change of alpha-helicity occurs in the first or second oxidation phase, although very slight changes in the helix parameters might be occurring. Concurrent with the destruction of tryptophan during the first phase is the appearance in CD difference spectra ([theta]NBS-treated fd - [theta]native fd) of positive maxima at 208-210 nm and negative maxima at 224 nm, with crossovers at 217 nm. Enormous difference ellipticities, per oxidized subunit of 50 amino acids, of +490,000 +/- 80,000 deg cm2 dmol-1 at 208 nm and -520,000 +/- 110,000 deg cm2 dmol-1 at 224 nm have been derived from the data.(ABSTRACT TRUNCATED AT 250 WORDS)


Fourier transform resonance Raman spectroscopy of phytochrome.

The Pr and Pfr forms of phytochrome in H2O and D2O have been studied by Fourier transform resonance Raman spectroscopy with near-infrared excitation (1064 nm). It is demonstrated that this technique is a powerful method for analyzing the chromophore structures of photosensitive pigments. The high spectral quality allows discussion of vibrational assignments based on an empirical approach using previously published data obtained from model compounds. The reduction in intensity of a high-frequency band assigned to the ring-C/D methine bridge vibration is an indication for the non-coplanarity of the ring D in Pfr. The high intensity of a C-H out-of-plane vibration also supports this hypothesis. In Pr, a broad peak at approximately 1100 cm-1 is assigned to an out-of-plane vibration of a strongly hydrogen-bonded pyrrole C=NH+ group. It is missing in Pfr, suggesting deprotonation of the corresponding ring during the transformation from Pr to Pfr.


Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer.

Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series.


Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.


Influence of chromatin folding on transcription initiation and elongation by RNA polymerase III.

Nucleosomes were assembled onto either closed circular plasmids containing a single Xenopus 5S RNA gene or a linear tandemly repeated array of Lytechinus 5S RNA genes. Both chromatin templates were found to vary in their extent of compaction, depending upon the type and concentration of cation in solution. Compaction of these chromatin templates led to a significant inhibition of both transcription initiation and elongation by RNA polymerase III. Thus, the transcriptional repression observed after incorporation of genes into chromatin depends not only on occlusion of the promoter elements through direct contact with histones but also on compaction of nucleosomal arrays which occurs under the conditions of the transcription reactions.


Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.

Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.


Alternative structures for alternating poly(dA-dT) tracts: the structure of the B-DNA decamer C-G-A-T-A-T-A-T-C-G.

The X-ray crystal structure of the decamer C-G-A-T-A-T-A-T-C-G has been solved with two contrasting cations, Ca2+ and Mg2+. Crystals with calcium are space group P2(1)2(1)2(1), cell dimensions a = 38.76 A, b = 40.06 A, and c = 33.73 A, and diffract to 1.7-A resolution. Crystals with magnesium have the same space group, cell dimensions a = 38.69 A, b = 39.56 A, and c = 33.64 A, and diffract to 2.0 A. Their structures were solved independently by molecular replacement, beginning with idealized Arnott B-DNA geometry. The calcium structure refined to R = 17.8% for the 3683 reflections greater than 2 sigma, with 404 DNA atoms, 95 solvent peaks, and 1 Ca(H2O)7(2+) ion. The magnesium structure refined to R = 16.5% for the 1852 reflections greater than 2 sigma, with 404 DNA atoms, 62 solvent peaks, and 1 Mg(H2O)6(2+) ion. The two structures are virtually identical and are isostructural with C-G-A-T-C-G-A-T-C-G [Grzeskowiak et al. (1991) J. Biol. Chem. 266, 8861-8883] and C-G-A-T-T-A-A-T-C-G [Quintana et al. (1992) J. Mol. Biol. 225, 375-395]. Comparison of C-G-A-T-A-T-A-T-C-G with C-G-C-A-T-A-T-A-T-G-C-G [Yoon et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6332-6336] shows that the expected alternation of twist angles is found in the central A-T-A-T-A-T region of the decamer (A-T small, T-A large), but the minor groove remains wide at the center, rather than narrow. Minor groove narrowing is produced, in these two structures, not by overwinding of the helix, but by an increase in base pair propeller. This analysis confirms the concept that poly(dA-dT).poly(dA-dT) is polymorphous, with different local conformations possible in different local environments.


Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.

To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.


T-T base mismatches enhance drug binding at the branch site in a four-arm DNA junction.

Base mismatches--non Watson-Crick pairing between bases--can arise in duplex DNA as a consequence of mutational events or by recombination. In a duplex, the sequence of the two bases involved, and those flanking the site of mismatch, determines the local structure and extent of destabilization of the helix. Base mismatches can arise also in recombination of nonhomologous strands, and their occurrence in Holliday recombination intermediates can influence the outcome of general or specialized recombination events. We have previously reported that the branch site in a DNA junction can interact selectively with a variety of ligands. Here we describe the thermodynamics of junctions containing T-T mismatches flanking the branch and show that these structures bind methidium and other intercalators with higher affinity than junctions lacking mismatches.


Linkage between ligand binding and control of tubulin conformation.

The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed; tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.


Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin.

Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Low-lying electronic states of carotenoids.

Four all-trans carotenoids, spheroidene, 3,4-dihydrospheroidene, 3,4,5,6-tetrahydrospheroidene, and 3,4,7,8-tetrahydrospheroidene, have been purified using HPLC techniques and analyzed using absorption, fluorescence and fluorescence excitation spectroscopy of room temperature solutions. This series of molecules, for which the extent of pi-electron conjugation decreases from 10 to seven carbon-carbon double bonds, exhibits a systematic crossover from S2----S0 (1(1)Bu----1(1)Ag) to S1----S0 (2(1)Ag----1(1)Ag) emission with decreasing chain length. Extrapolation of the S1----S0 transition energies indicates that the 2(1)Ag states of longer carotenoids have considerably lower energies than previously thought. The energies of the S1 states of spheroidenes and other long carotenoids are correlated with the S1 energies of their chlorophyll partners in antenna complexes of photosynthetic systems. Implications for energy transfer in photosynthetic antenna are discussed.


Bidirectional transport of spermine in rat liver mitochondria.

Further study of the mitochondrial transport of spermine (Toninello et al. (1988) J. Biol. Chem. 263, 19407) shows that, after loading rat liver mitochondria with [14C]spermine and [32P]phosphate, these components are released together into the surrounding medium by adding mersalyl or N-ethylmaleimide. On later addition of dithioerythritol, both are recaptured, but if acetate or nigericin are added instead, only spermine re-enters and there is continued export of phosphate. This bidirectional transport of spermine in and out mitochondria is driven, respectively, by membrane potential and pH gradient at constant protonmotive force. Results using [14C]spermine or [32P]phosphate, in conjunction with the their unlabelled isomers and with or without carbonyl cyanide/p-trifuloromethoxyphenylhydrazone (FCCP) present suggest that there is a continuous energy-dependent efflux-influx cycling of spermine and phosphate.


What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.


Sterically stabilized liposomes.

Many recent reports have demonstrated that rapid uptake of liposomes in vivo by cells of the mononuclear phagocytic system (MPS), which has restricted their therapeutic utility, can be overcome by incorporation of lipids derivatized with the hydrophilic polymer polyethylene glycol (PEG). The structure-function relationship of PEG-derivatized phosphatidylethanolamine (PEG-PE) has been examined by measurement of blood lifetime and tissue distribution in both mice and rats. The results are reviewed and contrasted with those from liposomes without PEG-PE or other surface modifications. With a PEG molecular weight in the range of 1000 to 5000, prolonged circulation and reduced MPS uptake is achieved. After 24 h, up to 35% of the injected dose remains in the blood and less than 10% is taken up by the two major organs of the MPS, liver and spleen, compared with 1% and up to 50%, respectively, for liposomes without PEG-PE. Other important advantages of PEG-PE have been identified: prolonged circulation is independent of liposome cholesterol content, degree of hydrocarbon chain saturation in either the PC or the PE lipid anchor, lipid dose, or addition of most other negatively charged lipids. This versatility in lipid composition and dose is important for controlling drug release in a liposome-based therapeutic agent. Steric stabilization has been proposed as a theoretical basis for the results and some initial results testing this hypothesis have been reported. A description of a theoretical model is presented here and evaluated with the data available. The results are compared with other particulate drug carriers and the range of potential applications are considered.


Detection and time-course analysis of phospholipid hydroperoxide in soybean seedlings after treatment with fungal elicitor, by chemiluminescence-HPLC assay.

Direct evidence for the existence of phosphatidylcholine hydroperoxide (PCOOH) and the determination of the changes in PCOOH after treatment of soybean seedlings with fungal elicitor, were examined using chemiluminescence-HPLC of hydroperoxide specific assay. PCOOH was transiently accumulated within 6 h after elicitor treatment but such an event was not observed in control plants, thus suggesting that accumulation of PCOOH was a response to fungal elicitor in the host plant. The result suggested that singlet oxygen had participated in the lipid hydroperoxidation, since non-conjugated fatty acid hydroperoxide isomers due to the singlet oxygen oxidation were detected in the phospholipid fraction. The reaction of lipid hydroperoxidation, preceding the induction of a defense compound, may be involved in the triggering of a plant defense mechanism against fungal invasion.


Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.


Amphipathic poly(ethylene glycol) 5000-stabilized dioleoylphosphatidylethanolamine liposomes accumulate in spleen.

Relatively small liposomes (d less than 200 nm) composed of dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) and containing dioleoyl-N-(monomethoxypoly(ethylene glycol)succinyl)phosphatidylethanolamine (PEG-PE), with PEG of M(r) 5000 (PEG5000-PE), accumulate in the spleen (approximately 40% i.v. injected dose), unlike dioleoylphosphatidylcholine (DOPC)/Chol/PEG5000-PE liposomes of similar size, which show prolonged circulation in the blood. Spleen accumulation was dependent on the injection dose, PEG-PE concentration, and the PEG chain length. The DOPE/Chol/PEG5000-PE liposomes are plasma stable and morphologically indistinguishable from DOPC/Chol/PEG5000-PE liposomes. These results reveal the significance of the matrix lipid in determining the circulation time of PEG-PE-containing liposomes, and are relevant to the design of liposomes which avoid or accumulate in the spleen.


Cholesteryl ester transfer protein promotes the association of HDL apolipoproteins A-I and A-II with LDL: potentiation by oleic acid.

The association of apolipoprotein (apo) A-I and apo A-II with apo-B-containing particles was measured after incubation at 37 degrees C of either total plasma or low-density lipoproteins (LDL) and high-density lipoproteins-3 (HDL3) in the presence of partially purified cholesteryl ester transfer protein (CETP). At the end of the incubation, apo-B-containing lipoproteins were separated by immunoprecipitation with an anti-apo B gamma-globulin fraction. In mixtures containing LDL and HDL3, either maintained at 4 degrees C or incubated at 37 degrees C, optimal concentrations of anti-apo B antibodies induced the precipitation of more than 95% of apo B without precipitation of apo A-I and apo A-II. When total plasma was incubated at 37 degrees C for 24 h, a significant proportion of apo A-I and apo A-II became associated with apo-B-containing lipoproteins. The fraction of HDL apoproteins associated with apo-B-containing lipoproteins was significantly reduced when plasma was supplemented with TP2 anti-CETP monoclonal antibodies, which are known to inhibit CETP activity. Incubation of LDL and HDL3 for 24 h at 37 degrees C in the presence of purified CETP also induced the association of a significant proportion of apo A-I and apo A-II with apo-B-containing particles. This effect was dependent on CETP concentration in the incubation mixtures and could be suppressed by the addition of anti-CETP monoclonal antibodies. While oleic acid alone, at a final concentration of 0.2 mmol/l, did not promote any association of HDL-apolipoproteins with LDL, it was able, at this concentration, to greatly enhance the CETP-mediated association of apo A-I and apo A-II with apo-B-containing particles. In the presence of both CETP and oleic acid, the association of apo A-I and apo A-II with apo-B-containing particles was apparent within 3 h of commencing the incubation. Approximately 3 mol of apo A-I and 1 mol of apo A-II co-precipitated with each mol of apo B after a 24 h incubation of LDL, HDL3 and CETP. When oleic acid was added to the incubation mixture in addition to CETP, up to 5.5 mol of apo A-I and 2.3 mol of apo A-II were associated with each mol of apo B.(ABSTRACT TRUNCATED AT 400 WORDS)


Antioxidant role of cellular reduced coenzyme Q homologs and alpha-tocopherol in free radical-induced injury of hepatocytes isolated from rats fed diets with different vitamin E contents.

Reduced coenzyme Q9 (CoQ9H2) and reduced coenzyme Q10 (CoQ10H2) as well as alpha-tocopherol (alpha-Toc) are known to be potent lipid-soluble antioxidants in mammalian tissues. Reduced coenzyme Q homolog (CoQnH2) appears to show antioxidant activity independent of that of alpha-Toc (Matsura, T., Yamada, K. and Kawasaki, T. (1992) Biochim. Biophys. Acta 1123, 309-315). To further confirm this, we have studied the antioxidant role of cellular CoQnH2 and alpha-Toc using hepatocytes isolated from rats fed diets containing deficient, sufficient, and excess amounts of vitamin E (VE). Cellular damage was induced with a hydrophilic radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The concentration of alpha-Toc in VE-deficient hepatocytes was approximately 1/12 that in VE-sufficient hepatocytes, whereas the concentration of alpha-Toc in VE-excess hepatocytes was approximately 7-fold that in VE-sufficient hepatocytes. The molar ratios of alpha-Toc to CoQnH2 (CoQ9H2 plus CoQ10H2) in VE-deficient, sufficient and excess cells were 0.03, 0.33 and 2, respectively. In the hepatocytes in these three dietary groups, alpha-Toc status had little effect on the concentration of CoQ homologs. These hepatocytes were incubated with 50 mM AAPH for 4 h. The cell viability in all groups of hepatocytes decreased rapidly after 3 h of AAPH treatment, and was associated with the increase of lipid peroxides. The loss of cell viability and the increase of lipid peroxidation in VE-deficient cells were more pronounced than those in the hepatocytes of the other two groups. The endogenous CoQ9H2 content of each group of hepatocytes decreased linearly with a reciprocal increase in oxidized CoQ9 after addition of AAPH, whereas the decrease of endogenous CoQ10H2 in each group during AAPH treatment was much less than that of endogenous CoQ9H2. alpha-Toc in the three VE dietary groups of hepatocytes was also consumed without a time lag after addition of AAPH, and it was not spared by CoQnH2, even in VE-deficient cells where the CoQnH2 concentration was 38-fold that of alpha-Toc. These results indicate that CoQnH2, especially. CoQ9H2, is a lipid-soluble antioxidant, which is as effective as alpha-Toc in rat hepatocytes under the conditions employed in this study, and acts independently of alpha-Toc to inhibit lipid peroxidation.


Albumin and fatty acid effects on the stimulated production of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) by human polymorphonuclear leukocytes.

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 microM and at a palmitic acid concentration of 100 microM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 microM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 microM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.


Binary phase behavior of angiotoxic oxidized cholesterols with cholesterol.

Binary phase diagrams of oxidized cholesterols with cholesterol were constructed in order to find a possible physical mechanism for these cytotoxic sterols as disruptors of cell membranes. Two compounds, 25-hydroxycholesterol and 7-ketocholesterol, behave similarly in solids with cholesterol, i.e., continuous solubility can be demonstrated over all concentrations with some tendency for individual molecular species to cluster in the liquid state. However, the two compounds have greatly different solubilities in phospholipid bilayers. The interaction of cholesterol with cholestane 3 beta, 5 alpha,6 beta-triol differs from that of the other two binaries since these molecules form a eutectic. This compound has been found to be soluble in phospholipid bilayers. Hence, from available evidence, the three oxidized cholesterols must employ different mechanisms for disruption of cell membranes, depending on which membrane component can most easily solubilize each.


Nucleotide sequence of a fragment of the rat tryptophan oxygenase gene showing high affinity to glucocorticoid receptor in vitro.

A nucleotide sequence (3.2 kb) of a DNA region located approximately between introns 4 and 7 of the rat tryptophan oxygenase gene was determined. Using filter binding studies and monoclonal antibodies against the glucocorticoid receptor a high affinity binding of this region to the glucocorticoid receptor was demonstrated.


Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.


Enriched sources of Escherichia coli replication proteins. The dnaG primase is a zinc metalloprotein.

Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis. The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage lambda PR and PL promoters in the pUC9-derived vector pCE30. Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx. 120,000 molecules per cell. The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture). Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 +/- 0.08 g atom of tightly-bound zinc per mol of protein. Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified. Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive.


Authentic temperature-regulation of a heat shock gene inserted into yeast on a high copy number vector. Influences of overexpression of HSP90 protein on high temperature growth and thermotolerance.

Heat shock protein HSP90 is relatively abundant in eukaryotic cells even in the absence of heat shock. Its precise function is still unclear, although it is apparently required in higher levels for growth at high temperatures. In this study Saccharomyces cerevisiae transformants were constructed with 50-150 copies of the homologous heat-inducible gene for HSP90 (HSP82) present on a high copy number episomal vector. These transformants were then used to demonstrate: (i) that this heat shock gene displays essentially normal regulation when present in yeast at high copy numbers; (ii) that yeast is an expression host suitable for the high level synthesis of HSP90; and (iii) that increasing normal cellular levels of HSP90 affects a number of physiological properties. The HSP82 gene is normally single-copy in the haploid yeast genome, yet even at 50 to 150 copies per cell it displayed almost normal basal and heat shock-induced levels of expression. Proper regulation of the heat shock element sequence controlling HSP82 is therefore not lost at high gene copy levels. In unstressed cultures in exponential growth at 25 degrees C the low basal expression of the multiple HSP82 gene copies caused a 3 to 7-fold HSP90 overproduction, but HSP90 levels increased 10-fold to 30-40% of total cell protein following temperature upshift to 39 degrees C for 75 min. Heat induction of the chromosomal genes for other heat shock proteins in the same cells was not suppressed relative to cells which were isogenic but for the possession of just a single HSP82 gene, this constituting further evidence that yeast can authentically regulate a large number of heat shock genes. HSP90 overproduction was not protective against heat killing, causing strain-dependent reductions in growth at 37.5 degrees C and in thermotolerance.


Isolation and characterization of a cdc 2 cDNA from Dictyostelium discoideum.

A cdc2 homologous sequence was amplified from Dictyostelium discoideum by the polymerase chain reaction and used to isolate several cDNA clones. The amino acid sequence encoded by these cDNAs exhibited approx. 60% identity to the Cdc2 proteins of other species. A cDNA containing the entire coding sequence complemented the temperature sensitive cdc28 mutant of Saccharomyces cerevisiae, although growth of the transformants was slow and limited. Southern blot analysis of restriction digests under high stringency conditions provided evidence that Dictyostelium contains a single cdc2 gene, although at lower stringency multiple fragments were detected, suggesting the existence of a cdc2 gene family. Northern blot analysis of RNA from different stages of Dictyostelium development showed that cdc2 mRNA levels increased during aggregation and then decreased to low levels by the pseudoplasmodial stage of development. By contrast, cdc2 mRNA levels remained relatively constant as cells passed from exponential growth to the stationary phase.


Electron microscopical projections of the large ribosomal subunit from Thermomyces lanuginosus.

Multivariate statistical analysis and hierarchical ascendant classification have been used to construct averages of two fundamental electron microscopical views of large ribosomal subunits from the thermophilic fungus Thermomyces lanuginosus. The first was roughly pentagonal in shape and corresponded to the canonical crown view seen in images of large subunits from prokaryotic species. The second and more prevalent projection was elliptical in shape, and by matching protuberances could be interpreted as the complex rotated from the crown orientation. Because of its ubiquity and consistency, this elongated view could potentially serve as the standard for structural comparisons of the large ribosomal subunit from eukaryotic organisms, and as the basis for a three-dimensional reconstruction.


Cloning and sequencing of a human cDNA from cytolytic NK/T cells with homology to yeast SEC7.

Using the technique of subtractive hybridization (natural killer (NK) minus T helper cell line Jurkat), a human cDNA clone named B2-1 was isolated and sequenced. B2-1 transcripts are highly expressed in NK and peripheral T cells, but not in the T helper cell line used in the subtraction. Two overlapping cDNA clones contained 3309 bp, in agreement with a predicted size of 3.2 kb obtained by Northern blot analysis. The cDNA contained a 5' open reading frame encoding a 398 amino acid (aa) protein with a calculated M(r) of 46,383. The protein contains N-linked glycosylation sites and a site for phosphorylation by protein kinase C. Database homology searches show that the deduced protein is novel and has three discrete domains with significant homology to several structural proteins. The most striking homology was to yeast SEC7 in the central domain of the gene (57% identical over 466 bp) and also the protein level (42% identical amino acids; 39% conserved amino acids). SEC7 is a large protein (2008 amino acids) found on Golgi vesicles that plays a role in protein transport.


The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.


The complete nucleotide sequence of the gene (rpoD1) encoding the principal sigma factor of the RNA polymerase from the cyanobacterium Synechococcus sp. strain PCC7942.

The complete nucleotide sequence of rpoD1 gene from Synechococcus PCC7942 has been determined. The nucleotide data have indicated the presence of an open reading frame of 1155 base pairs encoding a polypeptide which shares the framework structure for principal sigma factors of eubacterial strains.


Nucleotide and primary amino acid sequence of porcine lactoferrin.

A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.


Sexual dysfunction in cancer patients.

As a result of the convergence of increased numbers of cancer survivors, heightened awareness of quality of life issues and improved methods of treatment with sex therapy, the problem of sexual dysfunction after cancer treatment is receiving increased attention. In this article, the recent literature is reviewed by tumor site, including breast, bladder, colorectal, gynecologic, testicular, prostate, and other malignancies. Limitations and advances in the available literature are discussed, and recommendations for future research and clinical work are made.


Supportive care in marrow transplantation.

Marrow transplantation is now an accepted form of therapy for patients with aplastic anemia, genetic diseases, and a variety of malignant diseases. Intensive chemotherapy and radiotherapy regimens are necessary to eradicate cancer cells and allow engraftment of the transplanted marrow. Patients undergoing such treatment suffer prolonged marrow aplasia and immunosuppression and experience significant nonmarrow toxicities. This requires maximum supportive care including hyperalimentation, fluid and blood transfusions, antibacterial and antiviral prophylaxis, and measures to reduce organ toxicities and accelerate engraftment. The use of drugs to suppress the production of antitumor necrosis factor-alpha has shown promise in reducing the nonmarrow toxicities of the conditioning regimen. Prophylactic antibiotics that reduce gastrointestinal colonization with aerobic bacteria yet preserve anaerobic flora may more effectively reduce not only systemic bacteria but fungal infections as well. The impact of infections due to cytomegalovirus has been reduced by more effective prevention in patients who are cytomegalovirus negative and reactivation in patients who are cytomegalovirus positive. The use of recombinant growth factors will significantly reduce morbidity after transplantation by reducing the period of marrow aplasia.


Bone metastases and tumor-induced hypercalcemia.

Tumor-induced hypercalcemia and tumor-induced osteolysis are essentially due to a marked increase in osteoclast-mediated bone resorption, although the kidneys play an important contributory role in the genesis of tumor-induced hypercalcemia. Parathyroid hormone-like protein plays an essential role in tumor-induced hypercalcemia, and maybe in tumor-induced osteolysis, but other factors could also be responsible for the osteoclast activation secondary to the neoplastic infiltration of the skeleton. Treatment of tumor-induced hypercalcemia essentially consists of volume repletion and administration of potent anti-osteolytic drugs. The bisphosphonate pamidronate is particularly useful for that matter and a dose of 1.0 to 1.5 mg/kg can normalize serum calcium in about 90% of hypercalcemic cancer patients. The apparently low response rate of bone metastases to systemic antineoplastic therapy seems to essentially reflect the relative insensitivity of our current methods for assessing response in tumor-induced osteolysis. Newly developed biochemical markers of bone turnover could be particularly useful for that matter. Bisphosphonates are the most potent of the available inhibitors of osteoclast activity. Prolonged administration of oral pamidronate could reduce by almost one half the complications of tumor-induced osteolysis, and repeated bisphosphonate infusions could induce a dramatic relief of bone pain in one third and a sclerosis of lytic lesions in one fourth of the cases. These data must, however, be confirmed in randomized, blinded trials and many questions remain unanswered concerning the optimal therapeutic schemes. Medical therapy of tumor-induced osteolysis by noncytotoxic means has nevertheless become a reality.


Pleural effusions.

Pleural effusions are a common development in patients with cancer. The optimal means of control are not defined, but systemic treatment is often effective in patients with drug-sensitive tumors. Local treatments advocated in the management of malignant pleural effusions differ in the means of drainage, the length of time pleural tubes are left in situ, and the type of intrapleural therapy administered. Randomized trials published in the past few months have suggested that bleomycin sulfate may be superior to tetracycline as a sclerosant and that mitoxantrone does not add to control of effusions compared with large-bore pleural tube alone placed at time of thoracoscopy.


Recent progress and current problems in management of invasive fungal infections in patients with neoplastic diseases.

Patients with neoplastic diseases are predisposed to develop invasive fungal infections as the result of impairments of host defense mechanisms due principally to pharmacologic immunosuppression resulting from intensive cytotoxic chemotherapy, ablative radiation therapy, and corticosteroids. Candida species, Aspergillus species, and emerging opportunistic fungal pathogens comprise the principal etiologic agents of opportunistic mycoses in cancer patients. This paper reviews the recent progress, particularly during the year of 1991, in management of invasive fungal infections and the current problems of invasive mycosis, which confront patients with neoplastic diseases.


Cytogenetic and experimental models.

The sarcomas, particularly those of soft tissue origin, often contain characteristic immunohistochemical and cytogenetic features that are of diagnostic relevance. Over the past year, novel findings have been reported that are likely to improve present sarcoma diagnostic capabilities. Findings of particular interest have been reported for Ewing's sarcoma, peripheral primitive neuroectodermal tumor, rhabdomyosarcoma, and clear cell sarcoma.


Osteosarcoma and other cancers of bone.

Because of great improvements in survival and reduction in amputations in patients with osteosarcoma, investigators in the various fields involved in diagnosis and care of these patients are making greater efforts to contribute to better knowledge of the disease and the effects of treatment. Recent developments include basic studies on genetic abnormalities, evaluation of the role of modern imaging in predicting chemo-induced tumor necrosis and in defining tumor margins, and research into new agents, with new combinations and schedules for increasing prognosis and reducing side effects. Major efforts are also being made by surgeons in the difficult field of reconstruction after bone resections.


Advances in the diagnosis and management of sarcomas.

Cytogenetic alterations that characterize different histologic subtypes of soft tissue sarcomas have been identified. In a few situations, more precise chromosomal mapping has allowed identification of certain genes that may be involved in the development or tumor progression of sarcomas. Careful family histories must be elicited in sarcoma patients. While "cancer families" are rarely identified when screening close relatives of sarcoma patients, the discovery of the currently known tumor suppressor gene syndromes associated with germ line retinoblastoma gene and p53 gene defects were made possible by their association with sarcomas. Optimal management of primary sarcomas includes function-sparing complete resection and radiotherapy. Innovative radiotherapy, utilizing radiation sensitizers or brachytherapy, may increase local control in patients with unresectable tumors. New drugs are needed. Epirubicin and other anthracycline analogues do have significant activity; however, no other novel drugs have documented efficacy. Dose intensity is being explored with sarcoma trials providing the "vehicle" to evaluate new cytokines. Several mechanisms of doxorubicin resistance have been identified in cell lines and fresh tumors, including alterations in glutathione peroxidase activity and MDR-1 gene expression. These observations need to be taken to the clinic.


Rhabdomyosarcoma and other soft tissue sarcomas of childhood.

Childhood rhabdomyosarcoma and the other soft tissue sarcomas of childhood are a heterogeneous group of tumors with many histologic subtypes, affecting multiple anatomic locations and requiring the care of subspecialists from a variety of disciplines. Epidemiologic studies of childhood sarcomas indicate that a familial predisposition to malignancy may contribute to tumorigenesis in a proportion of patients. Diagnosis of these tumors may be difficult; immunohistochemical and cytogenetic analysis is clearly important. Large cooperative group studies have answered important treatment questions in past years; these studies are now being analyzed for clues to effective treatment of specific histologic and anatomic subgroups. The Intergroup Rhabdomyosarcoma Study IV is now open and the questions addressed are reviewed. New developments in late effects of therapy for childhood sarcoma are considered.


Ewing's sarcoma and extracranial primitive neuroectodermal tumors.

Ewing's sarcoma and primitive neuroectodermal tumor of bone and soft tissue are rare tumors, considered with the small round blue cell tumors of childhood. Recent advances link Ewing's sarcoma and primitive neuroectodermal tumor, and support the concept that these tumors represent stages of differentiation in a neoplasm of neuroectodermal origin. Advances in chemotherapy have resulted in the survival of the majority of patients presenting with these tumors. This review briefly outlines the year's literature regarding the histogenesis, biology, and treatment of this tumor.


Histology, histochemistry, tumor and serum markers in gastrointestinal tract cancer.

Integration of morphologic and molecular information is being pursued as a result of more extensive use of in situ hybridization techniques. Oncogenes, gene products, and viral genomes involved in the genesis and progression of gastrointestinal tumors have been located in specific histotypes or tumor sectors. The main advances in this area relate to cell-matrix interactions and the biologic mechanisms of tumor invasion. Some aspects of the morphogenetic sequences of gastric and colorectal adenocarcinomas and hepatocellular carcinoma have been elucidated. Studies of DNA ploidy have furnished results consonant with carcinogenesis models envisaging sequential genome alterations due to genetic instability of the transformed cell. The prognostic role of DNA content in gastrointestinal tumors, however, has not been unequivocally established. Research on tumor markers has been directed toward the tissue expression and distribution of apomucins with different molecular structures. Evaluation of tumor markers in body fluids has proved inadequate for the early detection and screening of gastrointestinal neoplasia. Some interesting results have been reported with regard to the monitoring of gastric and colorectal carcinomas by means of serum tumor markers.


The genetics, epidemiology, and early detection of gastrointestinal cancers.

Current recommendations for screening large populations for colorectal neoplasia have been promulgated by a number of researchers and authorities who generally agree that ongoing screening is justifiable in high-risk groups but not yet in average-risk groups. Nonetheless, it is thought to be justifiable to provide screening for average-risk individuals upon request. Choice of tools for screening remains under discussion. Colonoscopy is generally agreed to be justifiable in those patients with the highest risk, ie, members of families with a clear inherited tendency to develop colorectal cancer or those with a personal history of colorectal neoplasia. There is currently no agreement concerning the recommended tools for those with a weaker family history (one or two affected relatives), but regular fecal occult blood testing with occasional limited endoscopic examination of the bowel is usually favored. The new immunochemical-based occult blood tests show great promise for improved sensitivity and specificity. The evidence of the association between Helicobacter pylori gastritis and gastric cancer has been strengthened by three studies that show that patients with gastric cancer are more likely to have had infection in the years (up to 20) prior to diagnosis. The relative risk for cancer when infected with H. pylori is 3.6 to 6, but many H. pylori-positive individuals do not develop gastric cancer and additional factors must be operative. Probably the most exciting development for gastroenterology in 1991 is the identification of the gene on chromosome 5, designated APC, which is responsible for familial adenomatous polyposis.(ABSTRACT TRUNCATED AT 250 WORDS)


Imaging of gastrointestinal malignancies.

Many advancements in the imaging of gastrointestinal malignancies have been seen in the past year. Endorectal ultrasound and magnetic resonance imaging with an endorectal surface coil allow for more accurate staging of the depth of bowel wall invasion by rectal carcinoma. Monoclonal antibody imaging may detect metastases not found by other modalities while computed tomography arterial portography and intraoperative ultrasound improve our ability to identify liver metastases. Endoscopic ultrasound is also useful in the preoperative assessment of esophageal cancer and pancreatic endocrine tumors.


Determination of moxonidine (BDF 5895) in plasma by gas chromatography-negative ion chemical ionization mass spectrometry.

For the measurement of the pharmacokinetic behaviour of moxonidine, 4-chloro-5-(2-imidazolin-2-yl-amino)-6-methoxy-2-methylpyrimidine, an extremely sensitive analytical method was needed. The GC-MS method developed is specific and reliably detects moxonidine plasma levels down to 40 pg ml-1. Using negative ion chemical ionization (NICI) the M- fragment of the ditrifluoromethyl benzamide derivative of moxonidine (m/z 721) and the [M-HCl]- fragment of the ditrifluoromethyl benzamide derivative of clonidine (internal standard, m/z 673) were monitored in the selected ion monitoring mode, ensuring a specific and sensitive detection of the compounds. The validation process carried out included assay precision, repeatability, linearity, accuracy, stability and estimation of the detection and determination limits. The plasma-level time-curves and pharmacokinetic parameters from two volunteers after oral administration of 0.2 mg moxonidine are presented and demonstrate the practicability of the method in, for example, clinical studies.


The assay and resolution of the beta-blocker atenolol from its related impurities in a tablet pharmaceutical dosage form.

For the complete resolution and determination of the beta-blocker atenolol and its manufacturing impurities a high-performance liquid chromatographic method is developed using structured optimization techniques. The method utilizes a 300 x 3.9 mm i.d. stainless steel column packed with mu Bondapak C18 and a mobile phase containing methanol-25 mM potassium dihydrogen orthophosphate containing 0.06% (v/v) dibutylamine (20:80, v/v) (pH 3.0). The flow rate of 1.0 ml min-1 is used and a detection wavelength of 226 nm. The linearity and repeatability are good for the present compound over the range 1.5-510.0 micrograms ml-1 (r greater than 0.99 and RSD 0.27%, n = 10). Application of the method to 50 and 100 mg tablets gave recoveries of 99% (w/w) and reproducibilities of (RSD) 1.1 and 0.52%, respectively (n = 5). The manufacturing impurities are found to be greater than 0.5% (w/w) of the atenolol peak.


On-line phase-transfer catalysed dansylation of phenolic compounds followed by normal-phase liquid chromatography with fluorescence detection.

A study of the on-line phase-transfer catalysed dansylation of phenolic compounds is presented. The extraction-dansylation is performed in the extraction coil of a home-made flow-injection extraction unit. After phase separation, the organic phase is fed to a normal-phase liquid chromatographic system with fluorescence detection. Ethynyloestradiol, oestradiol and paracetamol are used as test compounds. The influence of temperature on the reaction is examined. Calibration graphs showed good linearity (r greater than 0.996) and limits of detection are satisfactory (8 x 10(-7) M for ethynyloestradiol, 2 x 10(-6) M for oestradiol and 5 x 10(-7) M for paracetamol). The method is not applicable for the assay of oxychinoline, phenylephrine and morphine.


A sensitive and reproducible HPLC assay for doxorubicin and pirarubicin.

A high-performance liquid chromatographic method with spectrofluorometric detection has been developed for the analysis of doxorubicin (DOX), pirarubicin (PIRA) and their metabolite, doxorubicinol, in plasma. The detection was performed at 480 nm for excitation, and 590 nm for emission. The proposed technique is selective, reliable, and sensitive. The limit of quantification was 2 ng ml-1 for DOX and 5 ng ml-1 for PIRA. The reproducibility of the analytical method through statistical coefficients is approximately 5%. The accuracy of the method is good; the relative error is less than 5%.


On-line dialysis, liquid chromatography and post-column reaction detection of oxytetracycline in salmon muscle extracts.

The development of a sensitive automated method for residue control of oxytetracycline (OTC) in salmon muscle is described. Tissue homogenate is dialysed and the dialysate enriched on a small on-line polystyrene column. OTC and the internal standard (tetracycline) are separated by HPLC on a polystyrene column using an ion-pair eluent system. The column effluent is mixed with sodium hydroxide and irradiated at 366 nm and the resulting derivatives monitored by means of a fluorescence detector (excitation: 358 nm, emission: 460 nm). By the method OTC is detected down to 5 ng g-1. The standard curve was linear (r = 0.9999) over the range 50-1000 ng g-1. Within-day and between-day relative standard deviations (n = 6) at 50 and 200 ng g-1 ranged from 1.0 to 1.7%.


Bicarbonate- versus lactate-based CAPD fluids: a biocompatibility study in rabbits.

Previous in vitro biocompatibility studies have shown bicarbonate-based continuous ambulatory peritoneal dialysis (CAPD) fluids to be superior to those based upon lactate/acetate. To evaluate these findings in vivo, 41 rabbits were subjected to CAPD for four weeks in a randomized prospective study using either Dianeal, a commercially available dialysis fluid containing lactate, or 87b, a bicarbonate-based CAPD fluid. Ten rabbits with CAPD catheters, which were flushed with a heparin solution every 36 hours, served as controls. None of the control rabbits showed clinical or histopathological signs of peritonitis, while 8 of 20 in the Dianeal group and 6 of 21 in the 87b group contracted peritonitis. Four rabbits in the Dianeal group had to be sacrificed early due to severe peritonitis. Post mortem examinations, including scanning and light microscopy, did not reveal any macroscopic or microscopic differences among the three groups of noninfected animals. No significant distinctions between the groups could be made for body temperature, weight gain, dialysate volume, dialysate differential leukocyte count, dialysate protein content, and food intake during the course of the study. In conclusion, the present animal model did not reveal any major difference in the biocompatibility between the lactate- and the bicarbonate-based CAPD fluids.


Peritoneal clearances in hypertensive CAPD patients after oral administration of clonidine, enalapril, and nifedipine.

The authors investigated whether the reduction of arterial pressure, induced by the oral administration of clonidine (CLO), enalapril (EN), and nifedipine (NIF), has any effect on peritoneal transport rates. The study was performed in nine hypertensive patients undergoing continuous ambulatory peritoneal dialysis (CAPD). The patients were submitted to administration of CLO, EN, and NIF, each in randomized succession for two weeks, after withdrawal of any hypotensive therapy for eight days (washout period). The nine patients underwent a four-hour dwell exchange using a 2.27 g/dL glucose two-liter bag after washout and after each hypotensive period. The following parameters were analyzed: mean arterial pressure (MAP), performed in the sitting position; net ultrafiltration; effluent/initial dialysate glucose ratio (GL D/Do); peritoneal clearance of K, BUN, creatinine (Cr), phosphate, beta-2 microglobulin (beta 2), total proteins, and the ratio between beta 2 and Cr clearance. Moreover, residual renal Cr and beta 2 clearances were analyzed. The three drugs significantly reduced MAP at a similar rate. The peritoneal transport parameters after CLO were similar to the results in the washout period. On the contrary, after EN and NIF therapy, Cr and beta 2 clearances were significantly increased, and GL D/Do decreased in comparison to the washout period. The other peritoneal transport parameters after EN and NIF were similar to the washout period. Residual renal Cr and beta 2 clearances after the three drugs were similar to those in the washout. these data suggest that after two weeks of therapy with EN and NIF, glucose, Cr, and beta 2 peritoneal transports are influenced by these hypotensive drugs irrespective of the effect on the arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Acquired renal cystic disease and its complications in continuous ambulatory peritoneal dialysis patients.

To determine whether there is any difference in the prevalence of acquired cystic disease and malignancy of the kidney in patients on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis. Relevant studies published from January 1983 to June 1991 were identified by manual search and MEDLINE search. We reviewed the studies in which acquired renal cystic disease and/or renal cell carcinoma developed in patients on CAPD. Details of the prevalence of acquired cystic disease and renal malignancy as a complication of acquired cysts in CAPD patients were tabulated with the duration of treatment. Acquired cystic disease was observed in 195 of 425 CAPD patients (41.1%), which is comparable to the prevalence of 47.1% (520/1103) seen in hemodialysis patients. The overall prevalence of renal cell carcinoma accompanying acquired cystic disease in this series of CAPD patients was 2 of 375 (0.4%), which is comparable to the prevalence of 1.5% (17/1103) in hemodialysis patients. So far, eight other instances of renal cell carcinoma complicating acquired cystic disease in CAPD patients were described as case reports. Retroperitoneal bleeding due to the rupture of acquired cysts has been reported on a few occasions. In this review no differences were detected in the prevalence or severity of acquired renal cystic disease in patients treated with CAPD as compared with those on hemodialysis. Therefore, the incidence of complications associated with acquired cysts may also be the same for the two treatment modalities, although reports on such complications are rare.


Weekly clearances of urea and creatinine on CAPD and NIPD.

Weekly creatinine clearance (Ccr) and weekly Curea/v (kt/v) are popular indices for quantitating the amount of peritoneal dialysis provided. Studies were undertaken on 44 patients on continuous ambulatory peritoneal dialysis (CAPD) and 10 patients on nightly intermittent peritoneal dialysis (NIPD) to compare relationships of weekly creatinine clearance to weekly urea clearance (Curea) divided by total body water (v). With a long cycle therapy such as CAPD, the ratio of weekly Ccr to weekly kt/v is higher than with a short cycle technique, such as NIPD, in the same patient. If patients are shifted from CAPD to NIPD maintaining the same weekly kt/v, the weekly Ccr will decrease. If patients are shifted from CAPD to NIPD maintaining the same weekly Ccr, then the weekly kt/v will increase. The clinical implications of these observations are unknown, but should be kept in mind for future studies comparing CAPD and NIPD.


Tidal peritoneal dialysis: preliminary experience.

To determine the feasibility of home tidal peritoneal dialysis (TPD) and to assess whether eight hours of TPD can achieve uremia control and urea removal equal to that of continuous cycling peritoneal dialysis (CCPD). An open enrollment pilot study. The Home Dialysis Training Center of the University of Iowa Hospitals and Clinics, a tertiary care teaching hospital. Nine patients experienced with CCPD and living 80 km to 280 km from the dialysis center began TPD, because they wished to decrease their dialysis time. Following baseline measurements, each patient was taught to perform TPD. TPD consisted of an initial fill volume of 40 mL/kg, a residual volume approximately 20 mL/kg, and tidal exchanges of 10 to 20 mL/kg to achieve the desired hourly flow rate. Clinic assessments took place every four to six weeks, and prescriptions were subsequently altered to attain urea removal equal to that of CCPD. Patient interviews were used to determine TPD acceptance. Prior to each clinic visit, dialysate effluent volume and dialysis duration were recorded, and a sterile sample of the effluent was obtained for urea, creatinine, and total nitrogen measurement. Urea and creatinine clearances increased with dialysate flow. Dialysate nonurea nitrogen was 3.0 +/- 0.2 mmol/kg/D and changed minimally with increasing dialysate volumes. Eight hours of TPD (initial fill: 40 mL/kg; residual volume: 20 mL/kg; tidal inflow: 20 mL/kg) with hourly tidal flow exceeding 40 mL/kg/hr and no daytime volume achieved urea removal equal to that of the patient's prior CCPD prescription. TPD can provide dialysis equal to that of CCPD within a shorter amount of time (eight vs ten hours), but uses a greater volume of dialysate (16.0 L for TPD vs 9.5 L for CCPD).


Comparison of exit-site infections in disconnect versus nondisconnect systems for peritoneal dialysis.

To determine if disconnect systems reduce the incidence of exit-site infections when compared to nondisconnect systems. We prospectively monitored exit-site infections and peritonitis rates in 96 disconnect patients (Y-set, automated peritoneal dialysis (APD)) and 60 nondisconnect patients (spike, ultraviolet connection device (UVXD)). A freestanding chronic peritoneal dialysis unit staffed by physicians from both a medical school and a private setting. All patients who began peritoneal dialysis at our unit were monitored, regardless of cause of end-stage renal disease (ESRD) or age. Patients were dialyzed using the system (Y-set, spike, etc.) most appropriate for their life-style and their ability to administer self-care. We attempted to follow disconnect and nondisconnect patients for a similar median time on dialysis and compared differences in exit-site infections. Peritonitis rates (episodes/pt year) were reduced for disconnect (0.60) versus nondisconnect (0.99) systems (p = 0.0006). Despite the marked reduction in peritonitis rates, there was no difference in exit-site infection rates (0.35 vs 0.38), the time to the first exit-site infection, or the time to the first catheter removal for disconnect versus nondisconnect groups. When individual systems were compared, differences in exit-site infection rates (episodes/pt years) were noted (0.62,spike; 0.26,UVXD; 0.32,Y-set; 0.41,APD). We found no overall difference in exit-site infection rates for disconnect versus nondisconnect systems, despite a reduction in peritonitis rates for disconnect systems.


Coexistence of essential thrombocythemia and multiple myeloma.

We describe a patient with essential thrombocythemia (ET) who developed multiple myeloma (MM) 5 years after the initial diagnosis. A review of the literature revealed no additional reports of the association of these two diseases. Development of MM was not related to treatment of essential thrombocythemia. This association suggests an alteration at the pluripotential stem cell level.


Elliptocytosis associated with an abnormal alpha glycophorin.

A case of elliptocytosis associated with an undescribed abnormal alpha glycophorin (alpha GP) is reported. Using immunoblotting techniques, a clear-cut minor band 6' was detected emerging just behind the monomer of delta GP (band 6) when probed with anti-alpha GP antiserum. It also reacted with anti-peptide C antiserum, suggesting that this new band with a molecular weight of 24 K is related to the structural alteration of alpha GP and not delta GP. The erythrocyte membrane proteins of the patient exhibited a quite normal pattern, with a normal alpha spectrin/beta spectrin ratio, but the reaction with anti-protein 4.1 serum confirmed the increase in proteolytic susceptibility of her protein 4.1. The results of DNA mapping implied that the abnormality may be due to a short deletion of the heterozygote. The significance of deviation involving the alpha GP and protein 4.1 to the elliptocytic change of erythrocyte shape is briefly discussed.


Blood transfusion in marrow graft recipients.

Of 182 consecutive patients undergoing allogeneic bone marrow transplantation (BMT), the relative numbers of those who received red blood cells (RBC), platelets (PLT), and granulocytes were 82%, 96%, and 26%, respectively. The transfused patients received an average of 1.26 (SD +/- 2.0) RBC units, 9.41 (SD +/- 13.2) PLT transfusions, and 0.33 (SD +/- 1.1) granulocyte concentrates per week per 50 kg body wt. in the period starting on the day of bone marrow transplantation (BMT) up to 60 days post BMT. The total number of units per transfused patient was 7.7 (range 1-63) RBC, 55.2 (range 2-394) PLT and 6.2 (range 1-36) granulocytes in the same period. Patients with grades II-IV acute graft-versus-host disease (GVHD) needed more RBC and PLT (p less than 0.001) than patients with grades 0-I acute GVHD. Patients with late engraftment required more granulocyte and PLT transfusion than those with early engraftment (p less than 0.05). Patients with high-risk malignancy had greater need for RBC and PLT than "low-risk patients" (p less than 0.02 and p less than 0.01), respectively). Patients with major ABO-incompatible donors showed a greater need for RBC than patients with minor ABO incompatibility (p = 0.02) or ABO identical donors (p = 0.01). Patients with relatively poor estimated survival required the most RBC and PLT.


Maintenance with low-dose cytarabine for acute myeloid leukemia in complete remission.

Thirty-four patients with acute myeloid leukemia (AML) in complete remission (CR), 30 of them aged over 60, received maintenance therapy scheduling four courses of low-dose cytarabine (LDA) 20 mg/m2/day in two subcutaneous injections for 3 weeks every 6 weeks. Each course was stopped when hematologic toxicity occurred, and doses of LDA were subsequently reduced by 50% for the following courses. During the first course of LDA, 15 patients needed blood and four patients platelet transfusions. Overall, 28 patients received four courses of LDA: 11 did not require any dose reduction, while 14 required one dose reduction and three needed two successive dose reductions. Two patients were hospitalized during maintenance. Median disease-free survival (DFS) is 308 days, with 16% of patients surviving at 5 years. Seven patients relapsed during the 168 days of maintenance, while ten of the 27 patients remaining at risk on day 169 relapsed during the 168 days following maintenance. We conclude that in AML in CR, the maximal dose of LDA tolerated by ambulatory patients is 10 mg/m2/day for 3 weeks. LDA seemed to delay relapse; however, precise assessment of the efficacy of this approach would require a randomized trial.


Anticardiolipin antibodies and lupus anticoagulant in patients treated with different methods of renal replacement therapy in comparison to patients with systemic lupus erythematosus.

Antiphospholipid antibodies were found to be associated with certain clinical manifestations such as recurrent venous thrombosis or arterial occlusions in a wide spectrum of immune disorders [6]. We analyzed the plasma concentration of two isotypes (IgG, IgM) of anticardiolipin antibodies (ACA) and lupus anticoagulant (LAC) activity in 84 patients with end-stage renal disease. They were receiving different types of renal replacement therapy and had a high frequency of thrombotic vascular complications. The prevalence of positive tests and the mean ACA concentration obtained in the plasma of renal patients were compared with those in patients with systemic lupus erythematosus (SLE) and in healthy controls. When analyzed as a whole group, renal patients maintained on dialysis (n = 45: hemodialysis, n = 20; peritoneal dialysis) or with a functioning kidney transplant (n = 19) did not differ in mean ACA concentration and LAC activity (n = 84, ACA: IgG 10 +/- 7 U/ml, IgM 2 +/- 1 U/ml, LAC ratio: 1.0 +/- 0.2) from healthy subjects (n = 50, ACA: IgG 10 +/- 3, IgM 2 +/- 1 U/ml, LAC ratio: 1.0 +/- 0.1) but they had a higher incidence of raised IgG-ACA titers (renal replacement 14% vs. normal controls 4%, p less than 0.05). No significant correlation was found between thrombotic events and raised ACA or LAC activity in dialysis patients. In contrast, the proportion of SLE patients (n = 51) with a raised concentration of ACA was significantly higher (IgG: 69%, IgM: 29%) than that among patients with renal replacement therapy (IgG: 14%, IgM: 4%) or normal controls (IgG: 4%, IgM: 2%, p less than 0.002). Moreover, recurrent manifestations of thrombosis in SLE were associated with very high IgG-ACA (n = 11, IgG 117 +/- 91 U/ml) and LAC activity (LAC ratio: 2.4 +/- 0.7) in comparison to SLE patients without thrombotic events (n = 40, ACA: IgG 23 +/- 13). The results of our investigations demonstrate that the pathogenetic role of these phospholipid antibodies in end-stage renal disease is far from established.


Monitoring prothrombin fragment 1 + 2 during initiation of oral anticoagulant therapy after intracoronary stenting.

Patients with intracoronary stent implantation are treated with aggressive anticoagulant and antiplatelet therapy consisting of high-dose heparin, phenprocoumon, acetylsalicylic acid, dipyridamole, and the infusion of dextran to prevent a subacute thrombotic occlusion of the stented segment. In an effort to optimize this treatment by reducing both imminent bleeding complications and subacute thrombotic occlusion, the concentrations of prothrombin fragment 1 + 2 (F 1 + 2) were determined after intracoronary Palmaz-Schatz stent implantation in 19 consecutive patients. The F 1 + 2 concentrations after stent implantation and before the initiation of oral anticoagulant therapy (OAT) were 0.35 nm/l and 0.25-0.53 nm/l (median and 25th-75th percentile), versus 0.74 nm/l and 0.52-0.78 nm/l, in healthy subjects and 0.61 nm/l and 0.30-1.02 nm/l in 15 patients with ongoing proximal DVT. Nine days after initiation of OAT, F 1 + 2 concentrations in both patient groups had not yet reached levels observed in patients with OAT in the stable state (0.16 nm/l, 0.12-0.26 nm/l; n = 76; P less than 0.0001 compared with healthy subjects; INR 2.0-4.5). Despite an INR greater than 2.0, accompanying heparinization was terminated on day 9. In two stented patients a minor bleeding complication arose after the removal of the arterial catheter. Subacute thrombotic occlusions were not observed. Since F 1 + 2 concentrations did not exceed the upper limit of normal range (1.11 nm/l) in any of the 19 patients, the therapeutic regimen was not changed. Monitoring F 1 + 2 may thus be helpful in introducing a more individual treatment if aggressive anticoagulation has to be performed.


Cytogenetics in multiple myeloma and plasma cell leukemia: simultaneous cytogenetic and cytologic studies in 51 patients.

Cytogenetic studies in patients with multiple myeloma (MM) and plasma cell leukemia (PCL) have in general been largely unsuccessful. The investigation of mitoses of nonmalignant hematopoietic precursor cells, rather than mitoses of malignant plasma cells might account for the low percentage of pathological genetic findings. We investigated bone marrow (BM) cells of 51 patients both cytogenetically and cytologically. In patients with a normal karyotype (n = 39) nearly all mitoses examined cytologically (107/117) derived from granulopoietic or erythropoietic cell lineages. In contrast, 20/27 metaphases in patients with a pathological karyotype (n = 12) were found to be plasma cell mitoses. These findings may explain the low rate of chromosomal rearrangements in MM and may suggest that the real abnormality rate is considerably higher.


Correction of neutropenia by GM-CSF in patients with a large granular lymphocyte proliferation.

The in vivo and in vitro effects of GM-CSF were tested in four patients with large granular lymphocyte proliferation (LGLP) and severe granulocytopenia. All patients had an increased percentage of LGL cells (greater than 20%), whereas 3/4 patients demonstrated rearranged T-cell-receptor genes. An effect on the peripheral granulocyte counts was noticed in 3/4 patients after 14 days of GM-CSF administration (5 micrograms/kg/day, subcutaneously); 2.5, 7, and 0.45 x 10(9)/l, respectively. These changes were associated with a two- to five-fold increase in monocytes and a strong increment in eosinophils. In one patient GM-CSF was not effective in increasing the granulocyte count. B lymphocytes showed a variable increase, ranging from 1.2-fold to 3.8-fold, while the number of NK cells remained almost constant during the GM-CSF treatment. No consistent effect of GM-CSF on T lymphocytes was observed. These data suggest that GM-CSF may be a therapeutic option in some patients with LGLP complicated by granulocytopenia and/or infection.


Iron status markers, serum ferritin and hemoglobin in 1359 Danish women in relation to menstruation, hormonal contraception, parity, and postmenopausal hormone treatment.

Iron status was assessed by measuring serum (S-) ferritin and hemoglobin (Hb) in a population survey comprising 1359 nonpregnant Danish women, in age cohorts of 30, 40, 50, and 60 years; 809 were premenopausal and 550 postmenopausal. Median age for menarche was 14 years, for menopause (artificial and natural) 48 years. Premenopausal women had lower S-ferritin (median 37 micrograms/l) than postmenopausal women (median 71 micrograms/l; p less than 0.0001). Of the premenopausal women, 17.7% had S-ferritin less than 15 micrograms/l (i.e., depleted iron stores), and 23.1% S-ferritin of 15-30 micrograms/l (i.e., small iron stores). Corresponding figures in postmenopausal women were 3.3% and 10.3%. Hb values in premenopausal women were mean 137 +/- 10 (SD) g/l (8.5 +/- 0.6 mmol/l) vs. 140 +/- 10 g/l (8.7 +/- 0.6 mmol/l) in postmenopausal women (p less than 0.0001); 4.1% of pre- and 3.3% of postmenopausal women had values less than 121 g/l (7.5 mmol/l). Iron deficiency anemia (i.e., S-ferritin less than 15 micrograms/l and Hb less than 121 g/l) was found in 2.6% of pre- and 0.36% of postmenopausal women. Premenopausal multipara had lower S-ferritin than nulli- and unipara (p less than 0.04). The use of oral contraceptives had a marked influence on iron stores; premenopausal women taking the pill had higher S-ferritin and a lower frequency of depleted iron reserves than nonusers (p less than 0.01). Postmenopausal estrogen treatment had no influence on S-ferritin or Hb.


Biosynthesis of guanidinoacetic acid in isolated renal tubules.

Guanidinoacetic acid, a precursor of creatine, is an essential substrate for muscle energy metabolism. Since guanidinoacetic acid has been reported to be synthesized from arginine and glycine by glycine amidinotransferase (transamidinase) in kidney homogenates or slices, the purpose of this study was to provide evidence of guanidinoacetic acid synthesis in isolated tubules from rat kidneys, and to clarify the mechanism regulating it. Isolated rat tubules were incubated with various substrates. Guanidinoacetic acid was separated by high performance liquid chromatography and measured fluorometrically. Results obtained were as follows: (1) Guanidinoacetic acid was synthesized from arginine or canavanine and glycine in isolated rat tubules. (2) D,L-Norvaline, ornithine and methionine suppressed guanidinoacetic acid synthesis. (3) Creatine suppressed guanidinoacetic acid synthesis, i.e. creatine was a negative feedback inhibitor of guanidinoacetic acid synthesis in this in vitro system. (4) Guanidinoacetic acid was not synthesized from hydroxyurea, citrulline, argininosuccinic acid or canaline. These data demonstrate that guanidinoacetic acid is synthesized only from arginine or canavanine and glycine, and that the guanidine cycle may not function fully in the rat renal tubule.


Comparison of the properties of ribonucleases in human liver tissue and serum.

Two ribonucleases (RNases) with acidic pH optima were partially purified, one from normal human liver tissue and the other from serum. The properties of the two enzymes were studied and compared. Liver RNase was partially purified about 700-fold by acid fractionation, phosphocellulose column chromatography, Sephadex G-75 gel filtration, and polyguanylate affinity column chromatography. Serum RNase was purified about 1200-fold by phosphocellulose column chromatography and Sephadex G-75 gel filtration. The two RNases showed a similar optimal pH and molecular mass, and similar behaviour towards metal ions, but they differed in their substrate specificity. Liver RNase displayed a higher activity towards polyuridylate (poly(U)) than towards polycytidylate (poly(C)), while serum RNase hydrolysed poly(C) more rapidly than poly(U). These findings suggest that liver RNase is not the primary source of the serum RNase with an acidic pH optimum.


Magnetic beads in suspension enable a rapid and sensitive immunodetection of human placental alkaline phosphatase.

The kinetics and efficiency of the interaction between placental alkaline phosphatase and a monoclonal antibody (laboratory number 327) were determined by immunoassay using microtitre plates or magnetic beads. While only up to 45% of placental alkaline phosphatase was bound to microwells precoated with this antibody, even after prolonged incubation, no less than 60% and 100% binding were reached using magnetic beads after 1 and 3 h incubations, respectively. High-molecular-mass placental alkaline phosphatase and complexed placental alkaline phosphatase forms were also completely bound to magnetic beads in the presence of deoxycholate (up to 9 g/l for serum samples). The assay sensitivity was improved up to 4-fold. In addition, 100% binding of the antigen was achieved during simultaneous incubation of magnetic beads, monoclonal antibody (125 micrograms/l), and placental alkaline phosphatase. This one-step enzymatic assay, based on magnetic beads, is an attractive alternative to the classic assay performed in microtitre plates, enabling rapid, precise, and sensitive antigen detection, and only necessitating a minimum of laboratory equipment.


Stability of blood gases, electrolytes and haemoglobin in heparinized whole blood samples: influence of the type of syringe.

The alterations of blood gases, pH, electrolytes and haemoglobin during 45 min storage in ice-water were measured in 6 types of syringes (1 glass and 5 plastic syringes, among these 3 "blood gas samplers"). It was confirmed that pO2 generally is not stable in plastic syringes. However, considerable differences among plastic syringes were found in this respect, the smallest increase occurring in an ordinary 2 ml syringe for injections and the greatest in one of the special blood gas samplers. Due to the "buffering effect" of deoxyhaemoglobin, the alterations of pO2 are smaller in the hypoxaemic than in the normoxaemic range. Relevant pO2 alterations in plastic syringes are demonstrable after 20 minutes. It is concluded that blood collected in plastic syringes must be analysed within 15 min after sampling, otherwise glass syringes should be used for blood collection. Deviations of pCO2, pH and electrolytes are described in detail. In general, they are due to sampling rather than to storage, and can be effectively minimized by a small dead space of the syringe and by use of an electrolyte-balanced heparin solution. The danger of erroneous haemoglobin measurements due to unequal resuspension of the red cells after storage is pointed out.


Performance characteristics of creatine kinase-MB isoenzyme measured with an immunoenzymometric and an immunoinhibition assay in acute myocardial infarction with and without thrombolytic therapy.

In a time study we compared the analytical and clinical performance of the Tandem Icon QSR CK-MB enzyme-immunoassay (Hybritech) (creatine kinase-MB) and a creatine kinase-MB immunoinhibition method (Boehringer Mannheim GmbH) (creatine kinase-B). Two hundred and ninety-nine serum samples from 38 patients suspected of acute myocardial infarction were collected at regular intervals during 48 hours. Twenty-nine patients were diagnosed as having acute myocardial infarction, of whom 19 received thrombolytic therapy. Although highly correlated, the large scatter around the regression line at low values indicated a different clinical performance of the two methods. We evaluated and compared test performance at different decision levels by means of frequency distributions and predictive values of positive and negative results. For early diagnosis of acute myocardial infarction (4 hours after onset of pain) the Hybritech creatine kinase-MB method gives acceptable predictive values. In thrombolytic treated acute myocardial infarction patients, the peak creatine kinase-MB and creatine kinase-B concentrations were reached after 13.0 h and 13.6 h after the onset of pain, compared with 19.8 h and 17.8 h for patients without thrombolytic therapy.


Determination of lipid hydroperoxides in serum iodometry and high performance liquid chromatography compared.

It is postulated that lipid peroxidation plays a role in the pathogenesis of a variety of diseases. Efforts have therefore been made to develop reliable and practicable procedures for quantifying lipid peroxidation products such as lipid hydroperoxides in biological specimens. An iodometric cholesterol colour reagent (Merck, Darmstadt, Germany) can be used to measure lipid hydroperoxides in isolated low density lipoproteins without lipid extraction. This method has been validated with respect to its analytical performance and suitability for serum samples by comparing it with a high performance liquid chromatography technique. The method was found to have acceptable performance characteristics with aqueous fatty acid hydroperoxide solutions (linoleic acid) and isolated low density lipoproteins, but it cannot be applied to native serum samples without extraction of lipids.


Temperature-dependent matrix effect in the direct enzymatic measurement of blood glucose.

The influence of the high-molecular-mass sample matrix in the direct enzymatic measurement of glucose in haemolysate was investigated by a comparison study using ultrafiltered haemolysate for reference. Haemolysate was obtained by 1:21 dilution of whole blood with a solution of digitonin and maleinimide. It was shown that at low protein concentration glucose distributes in a 1:1 ratio during ultrafiltration. With a hexokinase/glucose-6-phosphate dehydrogenase procedure excellent agreement was found between values measured in haemolysate (y) and ultrafiltrates (x), when incubation was performed at 25 degrees C (a = 0.047 mmol/l; b = 0.99; r = 0.999, n = 37); at 37 degrees C, however, the same procedure resulted in a non-tolerable systematic deviation in the direct analysis of haemolysate (a = -0.426 mmol/l; b = 1.00, r = 0.997, n = 37). The precision of measurements in haemolysate and ultrafiltrate was similar (CV 1.0-1.2%). Since stable reference material with an appropriate matrix is not available, it is important to evaluate haemolysate procedures carefully by comparison studies with patient samples. For reduction of experimental error in such studies we recommend the use of ultrafiltered haemolysate, since this can be analysed side by side with haemolysate in the same run.


Decreased serum homocysteine in pregnancy.

Total serum homocysteine was found to be significantly decreased in the first trimester of pregnancy, became maximal in the second trimester (about 50% compared with non-pregnant women), whereafter the concentration remained stable throughout the remainder of pregnancy. A return to normal concentrations was seen within 2-4 days post partum. Possible reasons for the decrease of serum homocysteine in pregnancy could be haemodilution and increased remethylation of homocysteine due to increased demands for methionine by the foetus.


Spectral analysis of EEG during self-paced movements: differences between untreated schizophrenics and normal controls.

Thirteen untreated schizophrenic patients, among them nine who had never been treated, were compared with a corresponding number of matched normal controls with regard to changes of the spectral composition of the electroencephalogram (EEG) accompanying voluntary movements. Triggered by self-paced movements of the right fingers (fast fist closure), the spectral composition of three epochs was analyzed: (1) rest (2,5-1,5 sec before movement), (2) movement preparation (last sec before movement onset), and (3) movement execution (1st sec following movement onset). For frequencies above 6 Hz, marked differences between schizophrenics and controls were evident, in particular over the parietal electrodes. Whereas patients exhibited a clear decrease of power density during movement as compared to rest, controls showed only a small decrease (left and mid parietal) or virtually none (only right parietal). Consequently there were significant differences over the right parietal area (P4) between patients and controls in the theta, alpha- and beta-bands with regard to the mean power density and center frequencies of these bands. Also at parietal positions, schizophrenics lacked the enhancement of theta-power during the preparatory epoch that was characteristic for normal controls at all parietal positions. The results are discussed with regard to the well-known disturbances of voluntary motor behavior in schizophrenia.


Basal sympathoadrenal function in posttraumatic distress disorder.

Research has consistently shown that patients with posttraumatic stress disorder (PTSD) manifest greater changes in heart rate, blood pressure, and plasma epinephrine than controls when exposed to trauma-related laboratory stressors. However, findings are equivocal as to whether PTSD subjects differ from controls on basal, or tonic, measures of autonomic activity. In this study, PTSD patients (n = 11) and asymptomatic controls (n = 11) were compared on measures of basal sympathoadrenal function, including plasma norepinephrine and epinephrine as well as heart rate and blood pressure. Results showed that PTSD patients were not significantly different from control subjects on any measure. Although phasic alterations in autonomic function in PTSD have been consistently found in previous research, this study suggests that tonic sympathetic nervous system activity in PTSD patients may not differ from that of healthy controls.


Effect of clomipramine and lithium on fenfluramine-induced hormone release in major depression.

Prolactin (PRL) and cortisol responses to oral administration of d-1 fenfluramine hydrochloride (60 mg) and placebo were examined in patients with endogenous major depressive disorder on three separate occasions: prior to treatment with clomipramine (CMI), after 4 weeks of CMI administration (175-250) mg/day), and 3 weeks after addition of lithium (Li) carbonate (serum level 0.5-0.9 mmol) to the treatment regimen. CMI significantly increased baseline PRL levels which were further elevated following Li supplementation. PRL response to fenfluramine (minus elevated baseline PRL levels) but not to placebo, was significantly increased by CMI administration, reflected over the 6-hr time course examined and in peak minus baseline values. Following addition of Li, the degree of enhancement was diminished although the peak minus baseline value remained significant relative to the pretreatment response. Cortisol levels were not increased by fenfluramine and were not altered by CMI or CMI + Li administration. The effect of CMI extends previous observations regarding the action of antidepressant treatment on serotenergically mediated hormone release. Methodological considerations relevant to the effect of CMI + Li are discussed.