Amyloidogenesis in Alzheimer's disease: basic biology and animal models.

A principal neuropathological hallmark of Alzheimer's disease is deposition of beta-amyloid, composed primarily of a 4 kD peptide, A beta. This peptide is derived from larger amyloid precursor proteins. The mechanisms that are responsible for A beta formation in vivo are unknown. Recently, transgenic strategies have been employed to test several hypothetical mechanisms in order to reproduce Alzheimer's disease-specific pathology in rodents.


The disordered neuronal cytoskeleton in Alzheimer's disease.

Evidence continues to accrue in support of the notion that normal adult human tau is converted into the protein subunits of Alzheimer's disease paired helical filaments as a result of the abnormal phosphorylation of tau at aberrant sites. Although the biological consequences of the generation of these abnormal tau derivatives in neurons remain uncertain, it is plausible that this process could destabilize microtubules and have a deleterious effect on the function and survival of neurons. Recent studies that probe the mechanisms whereby normal tau, a component of the neuronal cytoskeleton, undergoes profound alterations to become paired helical filaments in the Alzheimer's diseased brain are discussed.


Role of excitotoxicity in human neurological disease.

An increasing body of evidence has implicated excitoxicity as a mechanism of neuronal death in both acute and chronic neurological diseases. A major recent advance has been the successful cloning and expression of the non-NMDA, NMDA, and metabotropic glutamate receptors. The cellular mechanisms responsible for cell death following activation of these receptors are still being clarified. A recent advance in conceptualizing excitotoxicity is the notion that a slow excitotoxic process may occur as a consequence of either a receptor abnormality or an impairment of energy metabolism. It is possible that such a mechanism may occur in neurodegenerative illnesses. Recent therapeutic studies have focused on glycine site antagonists and on the efficacy of non-NMDA antagonists in ischemia.


Cytokines in neural regeneration.

Growth factors with already established multiple effects on non-neural cells continue to be of considerable interest to researchers with regard to the nervous system, where regulation of cell maintenance and plasticity in relation to lesion and regeneration is part of their functional repertoire. Fibroblast growth factors, interleukins, and type beta transforming growth factors are prominent representatives of such proteins. Ciliary neurotrophic factor is another multifunctional neurokine. The proposed role of this molecule as a 'lesion factor', however, is still not firmly settled.


Macrophages and nerve regeneration.

Macrophages are not only phagocytic cells but also secrete a plethora of growth factors that are potentially important for regeneration. This review will examine the emerging evidence of a likely contribution by macrophages to axonal regeneration.


Dopaminergic transplants in experimental parkinsonism: cellular mechanisms of graft-induced functional recovery.

The ability of intrastriatal grafts of fetal mesencephalic dopamine neurons to ameliorate the symptoms of experimental and clinical parkinsonism has raised the question of the mechanisms underlying the transplant-induced functional effects. Recent studies have taken advantage of quantitative cytochemical and in situ hybridization techniques to study functional graft-host interactions at the cellular level in the rat Parkinson model. The results provide evidence that behaviorally functional grafts restore dopaminergic neurotransmission and normalize dopamine receptor function in the denervated striatum, and that these effects are likely to depend on both synaptic and extrasynaptic mechanisms.


Synthesis of O-glycopyranosyl-N-hydroxysuccinimides of glucose and lactose and their opening by nucleophiles into prespacer glycosides.

O-Glycopyranosyl-N-hydroxysuccinimides of glucose and lactose have been synthesized. Peracetylated sugars, acetobromo sugars and benzoylated thioethyl glycosides have been used as precursors. With boron trifluoride etherate in dichloromethane, peracetylated sugars gave predominantly the beta anomer. Trimethylsilyl trifluoromethanesulfonate in nitromethane, however, gave predominantly the alpha anomer. Treatment of the acetylated O-glucopyranosyl-N-hydroxysuccinimide with various nucleophiles (methoxide, hydroxide and pentylamine) gave the corresponding deacetylated N-(succinyl)glucopyranosylhydroxylamines in almost quantitative yield. These derivatives are suitable for the formation of glycoconjugates or attachment of carbohydrates to solid phases using the opened succinimide as linking arm.


Determination of sequence and linkage of tissue oligosaccharides in caprine beta-mannosidosis by fast atom bombardment, collisionally activated dissociation tandem mass spectrometry.

Fast atom bombardment, collisionally activated dissociation tandem mass spectrometry (FAB-CAD-MS/MS), combined with p-aminobenzoic acid ethyl ester (ABEE) derivatization, were used to confirm the sequence and linkage pattern of subnanomolar amounts of the previously characterized three major thyroid gland oligosaccharides accumulated in caprine beta-mannosidosis. Positive ion FAB-CAD-MS/MS of both the [M + H]+ and [M + Na]+ ions from the ABEE derivatized oligosaccharides produced product ions derived from cleavage of the glycosidic bonds which allowed the sequences to be determined. Several fragments resulting from cleavages across the sugar ring permitted the assignment, in some cases, of the linkage positions between the sugar residues. The natriated molecule yielded several fragments of this type which were not observed when the protonated molecule was selected as the precursor ion. Use of these techniques gave the complete sequence and linkage characterization of the disaccharide and complete sequence and partial linkage information for the two higher oligosaccharides.


Fine sugar specificity of the Butea frondosa seed lectin.

Various monosaccharides and oligosaccharides were used to define the specificity of the Butea frondosa lectin using the hapten inhibition technique of human erythrocyte agglutination. Although B. frondosa lectin exhibited higher affinity for N-acetylgalactosamine, lactose and N-acetyllactosamine appeared to be relatively good inhibitors of haemagglutination. The behaviour of N-acetyllactosamine-type oligosaccharides and glycopeptides on a column of B. frondosa lectin immobilized on Sepharose 4B showed that the sugar-binding specificity of the lectin is directed towards unmasked N-acetyllactosamine sequences. Substitution of these N-acetyllactosamine sequences by sialic acid residues completely abolished the affinity of the lectin for the saccharides. The presence of one or several alpha Fuc(1-3)GlcNAc groups completely inhibited the interaction between the glycopeptides and the lectin. Substitution of the core beta-mannose residue by an additional bisecting beta(1-4)GlcNAc residue decreases the affinity of the lectin for these structures as compared with the unsubstituted ones.


Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies.

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this 'artificial' Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc alpha- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.


Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum.

Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.


Glycopeptidolipids--a new class of artificial antigens with carbohydrate determinants. Synthesis of artificial antigen with type-specific oligosaccharide hapten from Neisseria meningitidis group B.

Synthetic lipopeptide N-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide from Escherichia coli lipoprotein, was coupled with an oligosaccharide hapten from Neisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate--the artificial antigen of a new type processing the type-specific microbial determinant.


Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides.

Oligosaccharides from hyaluronic acid and chondroitin 6-sulfate were prepared by digestion with testicular hyaluronidase and separated according to their degree of polymerization by gel-permeation chromatography. These materials were successively analyzed by negative-mode ion-spray mass spectrometry with an atmospheric-pressure ion source. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of molecular species carrying multiple charges. Using two adjacent multiply charged molecular ions, the exact molecular weights up to the tetradecasaccharide were calculated with a precision of +/- 1 dalton. This type of mass spectrometry was also demonstrated to be feasible for the analysis of mixtures of oligosaccharides, including tetra-, hexa-, octa- and decasaccharides, from hyaluronic acid or chondroitin 6-sulfate without separation. Ion-spray mass spectrometry was thus shown to be applicable to the structural analysis of oligosaccharides from glycosaminoglycans.


Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.


Peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).


Surface glycoprotein of human natural killer cells recognized by wheat germ agglutinin.

We analyzed surface glycoproteins of human natural killer (NK) cells by utilizing lectins. Among the lectins tested, wheat germ agglutinin (WGA) was found to bind preferentially to CD16(Leu11)-positive lymphocytes as determined by two-colour flow cytometry. Analysis of glycoproteins in the lysate prepared from NK cells with sodium dodecyl sulfate (SDS) gel electrophoresis followed by Western blotting and 125I labeled WGA staining revealed that a glycoprotein with an M(r) of 65 kDa was strongly bound to the lectin, but no corresponding glycoprotein was detected in the lysate of T lymphocytes. This glycoprotein (GP65) gave several spots in the pI range 4.1-4.6 on 2-dimensional gel electrophoresis. Sialidase treatment of GP65 resulted in a single spot on the 2-dimensional gel, suggesting that GP65 is heterogeneous in the degree of sialylation. GP65 was shown to be exposed on the cell surface, since it was radiolabeled with 125I by the lactoperoxidase-catalyzed method. We next isolated GP65 from human peripheral blood lymphocytes by a combination of chromatography on a cation-exchange column and a WGA-agarose column and preparative SDS gel electrophoresis. It is suggested that GP65 is a novel surface glycoprotein on human NK cells.


Specificity of Amaranthus leucocarpus lectin.

We have demonstrated that Amaranthus leucocarpus lectin hemagglutinating activity was powerfully inhibited by the T-antigen, containing Gal(beta 1-3)GalNAc(alpha 1-3)Ser/Thr, and the Tn-antigen, which contains GalNAc(alpha 1-3)Ser/Thr. This suggests that the acetamido group at C-2 and the axial -OH at C-4 of the N-acetyl-D-galactopyranosylamine ring are important for lectin binding. The hemagglutination assays also established that desialylated and Pronase-treated human type O erythrocytes with an M phenotype were better recognized than erythrocytes from all other blood groups. The recognition was dependent on pH and ionic strength.


Comparison of the carbohydrate moieties of recombinant soluble Fc epsilon receptor (sFc epsilon RII/sCD23) expressed in Saccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells.

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFc epsilon RII/sCD23) was produced in Saccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFc epsilon RII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(alpha 2-3)Gal(beta 1-3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only alpha-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.


Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B.

The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11-76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1.A. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.


Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins.

Two new protocols for the three-dimensional, triple resonance, constant-time HCA(CO)N NMR experiment are presented that significantly increase the experimental resolution attainable in the C alpha frequency dimension. Experimental verification of the new experiments is provided by spectra of the IIA domain of glucose permease from Bacillus subtilis.


Toxin III of the scorpion Androctonus australis Hector: proton nuclear magnetic resonance assignments and secondary structure.

1H NMR has been applied to a 3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpion Androctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20-28) which is linked by two disulfide bridges to the central strand of a triple-stranded anti-parallel beta-sheet. Turns were identified at residues 15-17, 47-49 and also at residues 51-53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu37, Leu23, Val47, Tyr14, Trp45 and Tyr5. The Trp45 and Tyr5 rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.


A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.

A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues). Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly 15N-labeled protein were observed with heteronuclear single-quantum coherence spectroscopy (HSQC). In order to resolve overlapping cross peaks, selective 15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr and/or Val) was carried out using appropriate E. coli mutant strains. By this procedure, all the backbone 1H-15N cross peaks were classified into amino acid types.


The solution conformation of sialyl-alpha (2----6)-lactose studied by modern NMR techniques and Monte Carlo simulations.

We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used [1H,1H] NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear [1H,13C] NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on 13C chemical shifts, and (d) long-range heteronuclear scalar couplings across glycosidic bonds. We have used this approach to study the trisaccharide sialyl-alpha (2----6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the alpha (2----6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with phi angles at -60 degrees or 180 degrees and of the 3 staggered rotamers of the omega angle with a predominant gt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.


Multidimensional 1H and 15N NMR investigation of glutamine-binding protein of Escherichia coli.

Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp220 residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of L-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H epsilon 2 of His156 refines the earlier model where this particular proton forms an intermolecular hydrogen bond to the delta-carbonyl of L-glutamine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8-9 amino acid residues at a time. This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25,000.


Sequence-specific resonance assignment and secondary structure of (1-71) bacterioopsin.

The conformation of chymotryptic fragment C2 of bacteriohodopsin (residues 1-71) was studied by 2D 1H NMR. The fragment was solubilized in a mixture of chloroform/methanol (1:1), 0.1 M LiClO4. Most of the resonances in 1H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in beta-positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed alpha-helical regions Pro8-Lys30 and Lys41-Leu62 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala2-Gly6) adopts the helical conformation stabilized by 3 hydrogen bonds.


The effect of selective deuteration on magnetization transfer in larger proteins.

The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of the E. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuterated trp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NHi-NHi+1 cross peaks in the alpha-helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.


A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

A 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C, 15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.


A new 3D HN(CA)HA experiment for obtaining fingerprint HN-Halpha peaks in 15N- and 13C-labeled proteins.

A new 3D 1H-15N-13C triple resonance experiment is presented that provides in-phase absorptive cross peaks between amide protons and alpha-protons of the same and the sequentially preceding residue. The experiment yields similar connectivities as those described previously by Montelione and Wagner (1990a) (J. Magn. Reson., 87, 183-188) and Kay et al. (1991) (J. Magn. Reson., 91, 84-92). However, the pulse sequence was designed to minimize the time that transverse coherence of the 13Calpha nucleus is present, since this nucleus has the shortest transverse relaxation time of all the nuclei involved in these experiments. This is achieved by using a coherence transfer pathway from 1HN to 15N, 13Calpha, 1Halpha and back to the 1HN. In the sequence described, transverse 13Calpha coherence is present only for a length of ca. 1/1J(Calpha-Halpha). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly 15N- and 13C-enriched T4 lysozyme.


Solution conformation of human big endothelin-1.

The solution conformation of human big endothelin-1, a 38-residue peptide which serves as the putative precursor to the potent vasoconstrictor endothelin-1 has been examined by 1H NMR. NOEs were utilized as distance restraints in the distance geometry program DSPACE to generate initial structures. Further refinement of these structures was accomplished through molecular mechanics/molecular dynamics in an iterative process involving the incorporation of stereospecific assignments of prochiral centers and the use of back-calculation of NOESY spectra. A family of structures consisting of a type II beta-turn for residues 5-8 and an alpha-helix extending from residues 9-16 constitute a well-defined region, as reflected by the atomic root-mean-square (RMS) difference of 1.56 A about the mean coordinate positions of the backbone atoms (N, C, C alpha and O). This core region (residues 1-15) is very similar to the core residues of endothelin-1 (Donlan, M. et al. (1991) J. Cell. Biochemistry, S15G, 85). While the evidence from NOESY and coupling constant data suggests that the C-terminal region, residues 17-34, is not a mixture of randomly distributed chain conformations, it is also not consistent with a single chain conformation. Under the conditions studied, residues 17-38 in human big endothelin-1 in water at pH 3.0 between 20-30 degrees C appear to be represented by a series of conformers in dynamic equilibrium.


1H and 15N resonance assignments and secondary structure of the human thioredoxin C62A, C69A, C73A mutant.

The complete assignment of 1H and 15N backbone resonances and near-complete 1H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional 15N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay, J.D. et al. (1991) Biochemistry, 30, 2685-2698]. An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.


Simulation of NOESY spectra of DNA segments: a new scaling procedure for iterative comparison of calculated and experimental NOE intensities.

A new algorithm for simulation of two-dimensional NOESY spectra of DNA segments has been developed. For any given structure, NOE intensities are calculated using the relaxation matrix approach and a new realistic procedure is suggested for 1:1 comparison of calculated and experimental intensities. The procedure involves a novel method for scaling of calculated NOE intensities to represent volumes of digitised cross peaks in NOESY spectra. A data base of fine structures of all the relevant cross peaks with Lorentzian line shapes and in-phase components, is generated in a digitised manner by two-dimensional Fourier transformation of simulated time domain data, assuming a total intensity of 1.0 for each of the cross peaks. With this procedure, it is shown that the integrated volumes of these digitised cross peaks above any given threshold scale exactly as the total intensity of the respective peaks. This procedure eliminates the repetitive generation of digitised cross peaks by two-dimensional Fourier transformation during the iterative process of structure alteration and NOE intensity calculation and thus enhances the speed of DNA structure optimization. Illustrative fits of experimental and calculated spectra obtained using the new procedure are shown.


Quantitative comparison of experimental and simulated NOE intensities: correlation with accuracy of oligonucleotide structure determination.

The relation between the match of experimental and simulated NOE intensities with the accuracy of structure determination of oligonucleotides has been investigated. A hypothetical experimental spectrum of the oligonucleotide d(CCAACGTTGG) from its known X-ray crystallographic structure (Privé, G.G. et al. (1991) J. Mol. Biol., 217, 177-199) has been generated with simplifying assumptions of single correlation time, leakage rate etc., and this spectrum has been simulated imposing various constraints in a manner as one would do in a real case. The hypothetical spectrum represents the case of an infinitely good experimental spectrum and therefore the study of quality of fit against quality of structure represents the limiting case of real situations. It has been shown that even with a limited number of NOEs, it is possible to approach the correct structure of the molecule by demanding a highly accurate fit between experimental and simulated NOE intensities. Distance geometry calculations have been used to probe the extent of structural degeneracies in the NOE intensity matching exercise.


Quantitative measurement of small through-hydrogen-bond and 'through-space' 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin from Pyrococcus furiosus.

A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin (M(r) approximately 5.4 kDa) from Pyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a 'through-space' coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to 199Hg are significantly larger and fall in the 0.4-4 Hz range.


Activity patterns in elementary and high school students exposed to oxidant pollution.

We investigated activity patterns of 17 elementary school students aged 10-12, and 19 high school students aged 13-17, in suburban Los Angeles during the oxidant pollution season. Individuals' relationships between ventilation rate (VR) and heart rate (HR) were "calibrated" in supervised outdoor walking/jogging. Log VR was consistently proportional to HR; although "calibrations" were limited by a restricted range of exercise, and possibly by artifact due to mouthpiece breathing, which may cause overestimation of VR at rest. Each subject then recorded activities in diaries, and recorded HR once per minute by wearing Heart Watches, over 3 days (Saturday-Monday). For each activity the subject estimated a breathing rate--slow (like slow walking), medium (like fast walking), or fast (like running). VR ranges for each breathing rate and activity type were estimated from HR recordings. High-school students' diaries showed their aggregate distribution of waking hours as 68% slow inside, 8% slow outside, 10% medium inside, 9% medium outside, 1.5% fast inside, 1.5% fast outside. Elementary students' distribution was 47% slow inside, 15% slow outside, 20% medium inside, 12% medium outside, 2.5% fast inside, 3.5% fast outside. Sleep occupied 38% of high-school students' and 40% of elementary students' time; HR were generally lower in sleep than in slow waking activity. High school students' mean VR estimates were 13 L/min for slow breathing, 18 for medium, and 23 for fast; elementary students' were 14 slow, 18 medium, and 19 fast. VR distributions were approximately lognormal. Maximum estimated VR were approximately 70 L/min in elementary and approximately 100 L/min in high school students. Compared to adults studied similarly, students reported more medium or fast breathing, and had equal or higher VR estimates during slow and medium breathing despite their smaller size. These results suggest that, relative to body size, young people inhale larger doses of outdoor air pollutants than adults.


Measurement of personal exposure to NO2 in Sweden--evaluation of a passive sampler.

A passive (filter badge) sampler for personal NO2 exposure measurements was tested in a laboratory setting (exposure chamber), and in the field--outdoors, during periods of high relative humidity (mean 85%) and low temperature (-5 to +10 degrees C), and indoors, in an ice-hockey arena--using chemiluminescence as a reference method. Parallel measurements of NO2 in the exposure chamber (concentration range 100-825 micrograms NO2/m3) for 15, 30, and 60 min sampling periods, showed good agreement between methods. The concentrations obtained with passive samplers were 78 to 122% (mean 94%, SD +/- 11, N = 39) of those obtained with chemiluminescence, using a sampling rate (K'OG) of 0.14 cm/sec. The detection limits were 320, 160, and 80 micrograms NO2/m3 for 15, 30, and 60 minutes of sampling, respectively. Outdoors (concentration range 15-102 micrograms NO2/m3, concentrations obtained with passive samplers were consistently lower than concentrations obtained with chemiluminescence (mean 79%, SD +/- 9.3%, range 61-95%, N = 25), using the K'OG of 0.14 of cm/sec (Passive samplerNO2 = 0.67ChemilumNO2 + 4.5). A better agreement between concentrations obtained with passive samplers and chemiluminescence was achieved with a K'OG of 0.11 cm/sec (mean 100%, SD +/- 12%, range 78-121%, Passive samplerNO2 = 0.84 ChemilumNO2 + 6.4). Indoors (concentration range 210-3895 micrograms NO2/m3), concentrations obtained with passive samplers were 70 to 113% (mean 90%, SD +/- 16%) of the concentrations obtained with chemiluminescence (Passive samplerNO2 = 1.00ChemilumNO2 - 93) using a K'OG of 0.10 cm/sec. Duplicate samples collected indoors N = 18) and outdoors (N = 31) showed a variability (coefficient of variation, or CV) of less than 6%. It was concluded that the passive sampler is useful for measuring personal daily exposure as well as peak exposure. It is necessary to determine sampling rates for various environmental conditions.


Anticonvulsants in the treatment of bipolar disorder.

In the last decade, a proliferation of research has emerged concerning the use of somatic treatments with anticonvulsant properties, e.g., carbamazepine, valproate, clonazepam, oxcarbazepine, and electroconvulsive therapy (ECT), for patients with bipolar disorder. A sufficient number of controlled studies have been conducted to allow critical review of the evidence supporting the efficacy of these treatments for acute bipolar manic and depressive episodes, as well as for the prevention of subsequent episodes. Further research is needed to establish the prophylactic efficacy of anticonvulsants as maintenance therapies and, perhaps most importantly, to provide clinical and biological predictors of response.


Elimination kinetics of volatile organics in humans using breath measurements.

During the past decade significant strides have been made toward understanding the sources and factors which lead to volatile organic chemical (VOC) exposure in the general population. Less is known, however, about the impact of low-level environmental exposure on human health. Investigations are underway in a number of laboratories in an effort to determine the uptake, distribution, metabolism, and elimination kinetics for VOCs in humans. We examined the elimination kinetics for the third phase for ten VOCs--1,1,-trichloroethane, trichloroethylene, tetrachloroethylene, benzene, toluene, m,p-xylenes, o-xylene, ethylbenzene, p-dichlorobenzene, and limonene--in human subjects. Subjects were exposed to a variety of common consumer products and breath samples were collected post-exposure while the subjects spent up to 10 hr in a clean air environment. VOCs from breath samples were collected into canisters or onto Tenax GC cartridges and analyzed by gas chromatography-mass spectrometry. Exponential modeling of the decay data was performed to obtain kinetic parameters. The half-lives for trichloroethylene and 1,1,1-trichloroethane were approximately 5 to 8 hr for the four subjects. In general, the magnitude and range of variability was larger for toluene, limonene, and p-dichlorobenzene than for the other VOCs; the elimination rate spanning a few hours to a day or two. Thus, VOCs exhibit relatively short residence times in the body relative to other halo-carbons, such as polychlorinated biphenyls and dioxins.


Regional cerebral glucose metabolism and attention in adults with a history of childhood autism.

Sixteen high-functioning adults with a history of childhood autism and 26 normal control subjects underwent [18F]fluoro-2-deoxyglucose positron-emission tomography to assess regional cerebral glucose metabolic rate (GMR). Autistic patients had a left > right anterior rectal gyrus asymmetry, as opposed to the normal right > left asymmetry in that region. Patients also showed low GMR in the left posterior putamen and high GMR in the right posterior calcarine cortex. Brain regions with GMR > 3 SD from the normal mean were more prevalent in patients than in control subjects. This variable pattern of abnormal activity is consistent with heterogeneous neurophysiological etiology; group differences in striatum and cortex may represent a final common pathway.


Airborne asbestos levels in buildings: maintenance worker and occupant exposures.

A substantial number of air samples have been collected during the past few years to measure airborne asbestos levels in buildings with asbestos-containing materials (ACM). These samples fall into two categories: (i) samples collected to measure the exposure of workers while they were engaged in routine maintenance and repair activities; and (ii) samples collected during normal building activity to measure prevalent levels in buildings. The measurements derived from these samples have been compiled and summarized to provide estimates of the airborne asbestos exposure of workers engaged in routine maintenance and repair work and by other building occupants. These data indicate that maintenance and repair workers in buildings with ACM, after accounting for the frequency and duration of these types of activities, have annual exposure levels ranging from a median value of 0.002 fibers per cubic centimeter (f/cc) per year to 0.02 f/cc per year at the 90th percentile. Building occupants not involved in maintenance and repair work experience average exposure levels ranging from 0.00003 f/cc to 0.0005 f/cc.


Neurologic outcome of patients with dorsolateral prefrontal leukotomy.

Few controlled studies of neurologic function in frontal leukotomy patients have been done. The authors compared neurologic examinations of patients who had bilateral leukotomies with those of psychiatric control subjects matched for age, diagnosis, and duration of illness. Cranial nerve dysfunction, abnormal involuntary movements, and primitive reflexes were common. No significant differences between the two patient groups were found. The leukotomized patients were less irritable than control subjects and had statistically higher seizure and death rates. Neurologic abnormalities are, thus, common in elderly chronic psychiatric patients. Surprisingly, patients with bilateral prefrontal leukotomy do not differ from well-matched psychiatric controls on most clinical tests of neurologic function.


Visual perceptual dysfunction in patients with schizophrenic and affective disorders versus control subjects.

Visual perception was evaluated with standard tests for 26 hospitalized patients with schizophrenia, 23 hospitalized patients with affective disorders, and 60 control subjects. Both patient groups differed significantly from the control group on low amplitude of accommodation, esophoria or exophoria, vergence duction suppression, and convergence and divergence recovery ductions. Only the affective group showed significantly reduced fusion at near distance, and only the schizophrenic group differed significantly from control subjects on disorganized left apex formation. These findings appear to confirm the presence of visual perceptual disorders that can cause disability in psychiatric patients, with important research and rehabilitation implications.


Assessment of exposure to radon decay products in realistic living conditions.

The development of an automated system for activity-weighted size distribution measurements now permits more complete exposure and dose assessment for indoor radon decay products. Exposures characterized by the semi-continuous measurement of activity-weighted size distributions of radon decay products were obtained over four test periods in three normally occupied houses, two of which were occupied by cigarette smokers. These measured activity size distributions were used to calculate exposure-dose conversion coefficients and the annual effective doses. These doses were found to be approximately two-fold higher than the values derived conventionally from the measured radon concentration, on the assumption that indoor exposure to 20 Bq m-3 radon gas concentration corresponds to an annual effective dose 1 mSv y-1. The degree to which aerosol-measurement-based dose estimates were higher than radon-gas-based estimates was found to be influenced if the study house occupant was a cigarette smoker. Reassessment of the measured PAEC-weighted radon progeny particle size distribution in terms of the classical "unattached" and "attached" modes yielded lower estimates of the exposure-effective dose conversion coefficient that were similar to the reference values derived from a recent study by the National Research Council. Thus, by not resolving the measured radon progeny PAEC that is associated with particles intermediate in size between the two classical radon progeny modes, the estimated annual effective doses were also found to be similar to the values derived conventionally from the measured radon gas concentration. It is concluded that the observed presence of 4 to 13% of the radon progeny PAEC in the size-range 1.5 to 15 nm diameter is a dosimetrically significant factor that appears to be commonplace in various home environments.


Neuropsychological discriminators of long-term inpatient or outpatient status in chronic schizophrenia.

This study investigates specific neuropsychological functions discriminating schizophrenic patients with hospitalization beyond 18 months from patients residing in the community without rehospitalization beyond 3 years. Twenty-six Research Diagnostic Criteria-diagnosed chronic schizophrenic outpatients and 26 inpatients matched for sex, ethnicity, socioeconomic status, and chronicity completed a comprehensive neuropsychological battery and responded to an inventory of psychopathology. Discriminant function analyses showed that measures of motor coordination, preservation, memory, and attention discriminated between patient groups. Bilateral simultaneous fine motor coordination was the most potent discriminator between patient groups, independent of psychopathology. Implications for treatment and research on course and outcome in schizophrenia are discussed.


Human exposure to pollutants in Poland.

Serious environmental problems caused by decades of mismanagement of Poland's natural resources have been brought to light in recent years. All environmental media--air, water, food, and soil--have been burdened with toxic chemicals. Some environmental problems are so severe that the sources of pollution and the mitigation techniques needed are obvious, requiring no further research, but rather common sense, monies, and determination to implement the necessary controls and mitigation procedures. This paper will not address these obvious cases. Rather it will address that spectrum of environmental problems which requires a better understanding of public health risk in order to develop effective risk management strategies. Because these problems are numerous and monies limited, policy makers will need to set priorities both for research projects and control options. Using environmental concentration data presently available from Poland (especially for air), the paper will estimate human exposures, will point out research and monitoring needs, and hopefully, will lend credence to the concept that environmental policies and risk reduction strategies will be most effective if the Total Human Exposure Concept is used as the guiding scientific principle in risk assessment and management programs.


Bipolar disorder in idiopathic dystonia: clinical features and possible neurobiology.

The authors report 5 patients with bipolar disorders in the context of primary idiopathic dystonia. Four patients had DSM-III-R bipolar disorder, mixed, and one had cyclothymic disorder as diagnosed using the Structured Clinical Interview for DSM-III-R (SCID). All cases of bipolar disorder manifested rapid cycling. Three patients with bipolar disorder experienced onset of this illness soon after the onset of cervicocranial dystonia (5 neck dystonia, 4 craniofacial, 2 brachial, 1 vocal cord, 1 thoracic). These cases apparently represent a first report of bipolar disorders in dystonia. Clinical management, relevant literature, and putative neurobiology are reviewed.


Pathological lying associated with thalamic dysfunction demonstrated by [99mTc]HMPAO SPECT.

The authors report a case in which pathological lying is associated with right hemithalamic dysfunction as shown by [99mTc]HMPAO SPECT brain scanning. This association has not been demonstrated previously and is noteworthy because it supports the hypothesized roles of the thalamus and associated brain regions in the modulation of behavior and cognition.


Neurologic signs in patients with paranoid and nonparanoid schizophrenia.

Previous research found that both persons with schizophrenia and their unaffected relatives had significantly more neurologic signs than normal controls. As hypothesized, in this study neurologic signs were significantly greater in patients with nonparanoid than paranoid schizophrenia; the difference was greatest for "hard" signs.


Dissociation of neurocognitive deficits in a monozygotic twin pair concordant for schizophrenia.

Cognitive impairment varies markedly among schizophrenic individuals, although the reason for this is unknown. A monozygotic twin pair concordant for schizophrenia was evaluated using electrophysiological and neurobehavioral measures. Each twin showed a distinctive pattern of deficits; furthermore, clinical course and electrophysiological activity were not consonant with social and neurobehavioral functioning. Results suggest complex interrelationships between neurocognitive functioning and clinical presentation in schizophrenia.


Prolonged postictal encephalopathy in two patients with clozapine-induced seizures.

Two patients with prolonged postictal encephalopathy lasting 63 and 72 hours, respectively, following seizures with clozapine are reported. Clozapine alters the EEG in a majority of patients treated, with seizure frequency as high as 5-10% in doses above 600 mg/d. Prolonged postictal encephalopathy following a clozapine-induced seizure has not been previously reported but may be an important side effect of this medication. Pharmacologic and clinical issues are discussed.


The effect of adding Br or Zn supplements to the dialysate on the concentrations of Br and Zn in the blood of hemodialysed patients.

The effect of adding Br or Zn supplementation to the dialysate on the concentrations of Br and Zn in the blood of hemodialysed patients, is investigated. Patients with end-stage renal failure on hemodialysis show an abnormal trace element pattern. Our patients showed lowered serum Br and Zn concentrations. Four patients were subjected to dialysates with varying Br content. The impact on their serum and packed cells concentrations was evaluated. The supplementation resulted in an increase in the concentrations in serum and in packed cells. The Br concentration in serum and packed cells closely followed the dialysate content. In order to restore the Zn concentration to the normal level a ZnCl2 solution was added to the dialysate of 4 other patients. Zn accumulated in the patient as a consequence of its diffusion against the concentration gradient.


Cadmium uptake by brush border membrane vesicles from the rabbit renal external cortex.

Brush border membrane vesicles (BBMV) isolated from the rabbit renal external cortex were used to investigate inorganic cadmium (Cd) uptake. BBMV were incubated during 1 to 60 min in a buffered solution containing 10 to 200 mumol/L Cd (CdCl2), and uptake was determined using a rapid filtration technique. Cd uptake by BBMV was time-and concentration-dependent. At the lowest concentration (10 mumol/L), Cd uptake was nearly complete (70% of the plateau value, which was obtained after 10 min) within 1 min. At the highest concentration (200 mumol/L), the percentage of Cd uptake during the first minute was smaller (50%) and a slower component of uptake appeared. From additional experiments where Cd-loaded BBMV were submitted to an osmotic shock or to variations of extravesicular osmolality, it was concluded that the uptake of Cd by BBMV resulted essentially from membrane binding and not from intravesicular accumulation. The change in pH from 7.4 to 5.8 under iso- or hypotonic conditions removed about 40% of Cd bound to the BBMV, indicating that Cd binding to the membrane is pH sensitive. Thus, binding to the brush border membrane constitutes an initial step for inorganic Cd entry into renal epithelial cells.


Zinc in lymphocytes--the assessment of zinc status in patients with Crohn's disease.

A group of 26 Crohn's disease patients was compared to a healthy control group with regard to their zinc status in plasma, urine, hair, and lymphocytes. A method for lymphocyte preparation, which was in part newly designed, was used; it proved to be very effective as to recovery and purity of the harvested cell fraction. Results of the study suggest that an accurate estimation of zinc status can be more reliably obtained by determination of lymphocyte zinc--provided the cell preparation described is used--than by using any other of the examined body compartments.


Health evaluation of an aerosol formed in the fumes from mild steel welding electrodes.

Welding is one of the processes which cause multivarious problems to health. It requires considerable attention because of its severe hazards. During welding very harmful fumes and heat are emitted which have adverse effects on the health of the welder and people working in his vicinity. In the present work microfibre glass filter paper (GF/A) was found to be the preferable media for sampling of the fumes from mild steel electric welding electrodes for the evaluation of the trace elemental composition of the fumes. Using neutron activation analysis considerable amounts of the most harmful elements such as Ba, Co, Cr, Sb, Rb, Sc, As and Fe were observed.


Trace elements in hydatid disease.

The serum and cyst fluid levels of selenium, zinc and copper were investigated both in patients and sheep with hydatid cysts. Results were compared with those of healthy subjects in both species. The serum values of Se, Zn and Cu in healthy subjects were 60.24 +/- 1.96 micrograms/L, 0.59 +/- 0.07 mg/L, and 1.01 +/- 0.08 mg/L, respectively. The corresponding serum values of the three trace elements in patients with hydatidosis were 41.30 +/- 1.94 micrograms/L, 1.27 +/- 0.08 mg/L, and 0.89 +/- 0.09 mg/L. These results show a decrease in Se and Zn, and a rise in Cu in sera of patients with hydatid disease. Similar results were obtained in infested sheep as related to the healthy control animals. Our results on the three trace elements were found to correlate well with the incidence of cirrhosis, malignant and infectious diseases and arthritis. Se was not detectable in cyst fluid, while Cu and Zn levels were found significantly lower than serum levels. Cu and Zn levels in cyst fluid showed no difference in both species. Variations in serum Cu, Zn and Se levels were insignificant with regard to the genus of the host and the fertile capacity of the hydatid cyst.


Problems associated with the use of exclusion-diffusion chromatography for identification of zinc ligands in human milk.

The effect of operating conditions used in gel filtration, i.e. the type of gel and elution buffer, on the qualitative and quantitative distribution of zinc in human milk was studied. The gels used were Sephadex G10 and G15, Biogel P2 and Trisacryl GF05. The three elution buffers used were 0.05 M Tris-HCl, pH = 7.4, 0.05 M Tris-acetate, pH = 7.4 and 0.1 M ammonium acetate, pH = 6.5. Ultrafiltration was used as an additional technique for the verification of a number of the results obtained. Zinc ligands in human milk are, possibly, serum albumin and citric acid. The results showed a gel effect, which was greater on the identification and quantitation of the low molecular-weight ligand (the percentage of zinc associated with citric acid was lower with Biogel P2). The elution buffer had a considerable effect, particularly when quantifying the protein-bound fraction. The percentage of zinc coeluted with proteins decreased when the elution buffer contained acetate ions. The combination of Sephadex G15 and 0.05 M Tris-HCl, pH = 7.4 was found to be the best compromise. Zinc distribution with this system was 35-44% of zinc coeluted with proteins and 18-22% with citric acid.


Concentration changes of electrolytes in serum of chronic chagasic myocardiopathic patients. Sodium, potassium, chloride, calcium, inorganic phosphorus and magnesium.

Sodium, potassium, chloride, calcium, inorganic phosphorus (Pi) and magnesium contents in the serum of 75 myocardiopathic chagasic patients together with that in 29 healthy controls were determined. Blood samples were taken by catheterization from the superior cava vein (SCV), coronary sinus (CS), pulmonary artery (PA) and a peripheral artery, usually the femoral (FA). The results show that the average serum sodium, potassium, chloride, calcium and PI were invariably different in most samples, except the levels of potassium in SCV and Pi levels in FA. The serum concentration of magnesium was significantly different only in the blood of CS. In general one can observe that in the chagasic patients the serum sodium, chloride and calcium tend to diminish while serum potassium, Pi and magnesium tend to increase, especially in CS blood. The coronary gradient indicates that sodium, chloride and calcium tend to deposit in heart tissues, whereas the other elements (potassium, magnesium and Pi) tend to be released from this tissue. The systemic and peripheric gradients suggest that the chemical elements under study tend either to deposit in/or be released from different tissues depending on the course of development of the chagasic myocardiopathy.


Inhibitory effects of isolated inositol phosphates on zinc absorption in humans.

Inositol hexa phosphate (phytic acid) forms poorly soluble complexes with zinc, resulting in reduced absorption. During food preparation and processing phytate is partly hydrolyzed to lower inositol phosphates which have other binding properties. Zinc absorption in humans was measured in 18 women (22-37 a) by use of extrinsic labelling of meals with 65Zn and measurement of whole body retention. Inositol phosphates--400 mumol of the hexa phosphate form (IP-6), 200 and 400 mumol of the penta phosphate form (IP-5) and 400 mumol of the tetra phosphate form (IP-4)--were added to white bread and served with 200 mL of milk. The zinc content of the meal was 22 mumol. The absorption of zinc (65Zn) from white bread with no detectable phytate content was 43.3 +/- 17.9% (mean +/- SD) and from the breads with added 400 mumol of IP-6: 14.3 +/- 3.2%; 200 mumol of IP-5: 27.1 +/- 5.3%; 400 mumol of IP-5: 18.1 +/- 4.2%; and 400 mumol of IP-4: 41.5 +/- 11.3%. The results indicate that IP-6 and IP-5 have zinc-absorption depressing properties, while IP-4 added in isolated form is relatively inert as regards zinc absorption. However, a comparison with earlier obtained results from zinc absorption studies indicate that results obtained with isolated inositol phosphates can not directly be extrapolated to the phytate contents of foods.


Monocyte mediated cytotoxic activity against melanoma.

The appearance of macrophages in human tumours, the so called tumour-infiltrating or tumour-associated macrophages (TIM or TAM) has suggested a role for these cells in host defence mechanisms against cancer. In this review we discuss monocyte-mediated cytotoxic activity against melanoma, as reported by a number of different authors. Although most studies described in this review have used melanoma cell lines as targets for monocyte/macrophage cytotoxicity, it would be incomplete if cytotoxicity against other target cells such as K562 or WEHI-164 is not discussed. At least two distinct mechanisms of killing by monocytes/macrophages can be distinguished; direct recognition and cytotoxicity by the effector cells and antibody-mediated lysis of the tumour cells (antibody-dependent cellular cytotoxicity, ADCC). Both types of cytotoxicity will be discussed.


Glutathione content of human malignant melanoma cell lines: correlation of flow cytometric and biochemical assays, and application to human tumour aspirates.

We measured the glutathione content of a panel of human malignant melanoma cell lines by flow cytometry after staining with mercury orange, using visible light (488 nm) excitation, and compared the values obtained with those measured biochemically using a modified Tietze assay. Glutathione levels were depleted by culturing cells with various concentrations of buthionine sulphoximine to provide a suitable spread of glutathione concentrations. The two assays showed good correlation (r = 0.93). We found a number of technical factors to be critically important. In particular, the conditions of staining, cell number, and method of mixing media, cells and stain were responsible for wide variations in fluorescence intensity. We applied the flow cytometric technique to cell suspensions obtained by fine needle aspiration biopsy of human malignant melanoma deposits, and observed a proportion of cells with high glutathione levels in many samples. The results confirm the feasibility of measuring glutathione content by visible light flow cytometry, and raise the possibility of monitoring glutathione content as an indicator of drug resistance in clinical therapy of human malignant melanoma.


Inactive cathepsin B-like enzyme in human melanoma culture medium.

An inactive cathepsin B-like enzyme with a molecular mass of 40 kD was found in a human melanoma culture medium. The inactive form of this enzyme was converted into an active form with a molecular mass of 28 kD by pepsin treatment. This activated cathepsin B-like enzyme had almost the same characteristics regarding molecular size, substrate specificity, dependence on chemical reagents, and Km values as intracellular cathepsin B. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by electroblotting with an antiserum against cathepsin B yielded inactive cathepsin B-like enzyme fractions which showed two immunoreactive bands with molecular masses of 40 and 28 kD, respectively. On the other hand, alkali treatment of the inactive cathepsin B-like enzyme fractions released a cysteine proteinase inhibitor with a molecular mass of 12 kD. These data suggest that these inactive cathepsin B-like enzymes in melanoma culture medium are present not only in the precursor form, but that they are also present as enzyme-inhibitor complexes, both of which can be activated enzymatically in vitro.


Multipoint linkage mapping of the Emery-Dreifuss muscular dystrophy gene.

The clinical features to establish the diagnosis of X-linked Emery-Dreifuss muscular dystrophy (EMD) were recently redefined at the European EMD workshop in Baarn 1991. These criteria were used to select families from the literature and two new families for linkage analysis with the DNA markers F9, DX52, DXS15, F8C and DXS115. Recombinations are observed with the DNA markers F9, DXS52 and DXS15. No recombinations were found with F8C and DXS115. Multipoint linkage analysis indicates with a maximum location score of 73.9 that the EMD locus maps very close to F8C.


Regional distribution of common and dysplastic naevi in relation to melanoma site and sun exposure. A case-control study.

The frequency of melanoma (CMM), and of common and dysplastic naevi (CN and DN) in areas of skin chronically, intermittently and rarely exposed to UV light was investigated in 121 melanoma patients (30-50 years) and 310 controls. Both cases and controls had significantly more CN in intermittently exposed areas than in areas chronically or rarely exposed. The ratio of observed to expected number of CMM was also highest in intermittently exposed skin (1.3 compared to 0.8 in chronically exposed and 0.5 in rarely exposed areas). Thus, intermittent UV exposure seems to have the most potent 'naevogenic' as well as carcinogenic effect on melanocytes. Nineteen per cent of controls and 56% of cases had naevi fulfilling the clinical criteria for DN. The distribution pattern of DN was clearly different from that of CN and does not accord with the idea that UV light is a major aetiological factor for DN. The probability of CMM significantly increased with the degree of relative clustering of CN (p less than 0.05) and of DN (p less than 0.01). This co-variation of naevi and CMM over the body surface might be the result of the local insults to the melanocyte system caused by UV light and/or to the fact that naevi are precursor lesions of CMM.


The usefulness of single and combined clinical characteristics for the diagnosis of dysplastic naevi.

A total of 761 melanocytic lesions were studied to elucidate the usefulness of clinical features for the diagnosis of dysplastic naevi. Characteristics associated with high (irregular border, irregular pigmentation), intermediate (black coloured areas, largest diameter greater than 0.5 cm, change of size, change of colour) and low diagnostic efficiency could be defined. Combinations of criteria had high sensitivities: at least one of the following four criteria was positive in 96% of the dysplastic naevi and in all melanomas with less pronounced clinical characteristics: irregular border, irregular pigmentation, greatest diameter greater than 0.5 cm, black coloured areas. A lesion is therefore unlikely to be a dysplastic naevus or a melanoma if all these criteria are absent. When change of size and change of colour were analysed in addition to the features mentioned above a sensitivity of 0.96 was found for at least two of these six criteria. At least three of these six criteria were observed in all melanomas with less pronounced clinical characteristics. However, a rather low specificity (0.19 for at least one of four positive criteria, 0.20 for at least two of six positive criteria) indicated that dysplastic and non-dysplastic naevi cannot be clinically differentiated with acceptable certainty. With less stringent histological criteria approximately twice as high specificities were found. Specificities were about twice as high in a subgroup of patients with at least one proven dysplastic naevus besides the lesion under diagnostic consideration. This facilitates the identification of individuals at risk of developing a melanoma.


A possible role for cysteine proteinase and its inhibitors in motility of malignant melanoma and other tumour cells.

The metastasis of malignant tumour cells depends on their rapid replication, and their ability to adhere to the matrix of a biological barrier such as basement membrane, to degrade the matrix, and to migrate through this more permeable barrier. Secreted enzymes, including the cysteine proteinases cathepsins B and L, are known to degrade basement membrane components. Using a barrier-free substratum we studied the possible role of cysteine proteinases in influencing the motility per se of metastatic cells. We found that stefins, the natural inhibitors of cysteine proteinases, markedly decreased the stimulated motility of both human melanoma cells and W256 carcinosarcoma cells at low concentrations (0.5 microM). A stefin also inhibited melanoma cell adherence, but to a lesser extent than motility. Additionally, synthetic inhibitors (E-64, diazomethyl ketones) of cysteine proteinases were found to depress stimulated motility of W256 cells. These results suggest that cysteine proteinases and their inhibitors may have a direct role in the development of a migratory response per se in tumour cells.


A deletion including the brain promoter of the Duchenne muscular dystrophy gene is not associated with mental retardation.

A total of 161 unrelated Duchenne (DMD) and Becker muscular dystrophy (BMD) patients were screened for deletions in the brain promoter region of the dystrophin gene. Southern blot analysis using a probe for the brain promoter detected a deletion in this region in only one of the DMD families, in a patient with normal intelligence. This deletion also included the promoter of the muscle-type dystrophin and the exons encoding the actin-binding and part of the spectrin-like domains. Our data suggest that deletions in the brain promoter region are rare in DMD and are compatible with normal intelligence.


Outpatient surgical treatment of cutaneous melanoma.

From January 1988 to December 1989, 920 patients with pigmented cutaneous lesions, clinically diagnosed as suspected or certain cutaneous melanoma (CM), underwent excision under local anaesthesia as outpatients. Histological examination confirmed CM in 135 patients. Patients in this group whose initial excision was for biopsy purposes only (extending 1-2 mm beyond the lesion margin) underwent a subsequent radical excision (extending greater than or equal to 10 mm from the neoplastic margin). The second resection was carried out within 10 to 15 days of the first, on an outpatient basis if the thickness of the CM was less than or equal to 2 mm, and in hospital if it was greater than 2 mm. The clinical diagnosis proved correct in 88 cases (65%) where exeresis was the definitive surgical treatment. Outpatient surgery seems to be the best method for easing a workload dominated by the need to examine a growing number of pigmented skin lesions, without altering the prognosis for CM.


Isomers of a marine diterpene distinguish sublines of human melanoma cells on the basis of apoptosis, cell cycle arrest and differentiation markers.

The action of the marine furanoditerpenes, spongiatriol (SP) and episopongiatriol (ESP), were compared in two sublines of human melanoma cells (MM96E and MM96L) derived from the same metastatic lesion. MM96E had higher tyrosinase activity and lower expression of alkaline phosphatase but was otherwise indistinguishable from MM96L. SP and ESP treatment of both cell lines for 72 h at cytostatic doses inhibited B8G3 expression and tyrosinase activity but had little effect on the expression of tyrosinase antigen. MM96L cells were affected more than MM96E. SP and ESP induced apoptosis in both cell lines, ESP causing dendritic morphology in a proportion of MM96L cells. SP induced a marked G2/M arrest in MM96E cells. SP and ESP together define subtle qualitative and quantitative differences in human melanoma phenoypes, possibly based on expression of a repertoire of neurotransmitter receptors.


Uptake of [131I]thiouracil in tumours of patients with disseminated malignant melanoma. A pilot study.

Previous studies on mice carrying melanoma have shown that 5-iodo-2-thiouracil (ITU) is accumulated in the tumours due to its specific incorporation into melanin during its synthesis. ITU is also selectively localized in murine melanoma metastases and in cultured human melanoma cells. Progressive formation of melanin is, however, a prerequisite for the incorporation. Four patients with disseminated melanoma were injected intravenously with 39-62 MBq [131I]TU. Blood and urine samples were gradually collected, and 3-7 days postinjection tumours were biopsied and examined by impulse counting. The patients were scanned with a gamma camera over the total body daily for 3-4 days. The radioactivity was rapidly excreted. Poor melanin pigmentation of the tumours and low proliferation rate (possibly induced by chemotherapy) decreased the uptake of radioactivity by the tumors, and no imaging was possible. One of the patients, however, had clearly progressive disease with darkly pigmented metastases which contained considerably higher levels of radioactivity than the surrounding skin. Calculations indicated that a doubling of the radioiodine dose would probably make visualization of the tumours possible.


Early onset autosomal dominant progressive muscular dystrophy presenting in childhood as a Becker phenotype--the importance of dystrophin and molecular genetic analysis.

We present two cases of autosomal dominant limb girdle muscular dystrophy in a father and son. Both presented in childhood with a classical Becker muscular dystrophy phenotype. The father had initially been informed that he would not have affected children. After the diagnosis of muscular dystrophy in the son, immunoblot analysis was performed on muscle and revealed normal dystrophin. The polymerase chain reaction did not show any deletions in the dystrophin gene, and the father's dystrophin gene was not passed to his son. These cases demonstrate that autosomal dominant muscular dystrophy may present in childhood, and that dystrophin and molecular genetic analyses should be performed when considering the diagnosis of childhood muscular dystrophy, even in the presence of a classical phenotype.


Thermal neutron capture therapy of malignant melanoma using 10B-monoclonal antibodies: in vitro and in vivo analysis.

We have established methods of targeting a sufficient number of 10B atoms on human melanoma cells to allow selective destruction of the cancer cells by thermal neutron irradiation. Thermal neutron capture therapy (NCT)1-3 requires the presence of at least 10(9) 10B atoms on each target cell for specific killing of that cell without injuring normal tissues. In order to accumulate an adequate number of 10B atoms on target cells, we first created an effective compound containing 12 atoms of 10B per molecule (10B12-chlorpromazine) and 10B-dopa analogue (10B1-paraboronophenylalanine). In the present study, about three molecules of our newly synthesized 10B12-compound were conjugated to an avidin molecule. The resulting 10B38.5-avidin compound can be specifically directed to human melanoma cells by biotinated monoclonal antibodies (MAbs) specific for the cells. We were able to accumulate 2.6 x 10(8) 10B atoms on a melanoma cell using this method. Cultured human melanoma cells treated with 10B-avidin-biotin-MAb (10B-AB-MAb) were selectively damaged by thermal neutron irradiation in vitro. This is the first study to indicate that thermal neutrons selectively damage target cells boronated by MAbs.


Melas: an original case and clinical criteria for diagnosis.

We describe the full history and postmortem findings in one of the first identified cases of mitochondrial encephalomyopathy with stroke-like episodes (MELAS). To clarify diagnostic criteria, we analyzed 69 reported cases. The syndrome should be suspected by the following three invariant criteria: (1) stroke-like episode before age 40 yr; (2) encephalopathy characterized by seizures, dementia, or both; and (3) lactic acidosis, ragged-red fibers (RRF), or both. The diagnosis may be considered secure if there are also at least two of the following: normal early development, recurrent headache, or recurrent vomiting. There are incomplete syndromes in relatives of patients with the full syndrome and incomplete syndromes might also be encountered in sporadic cases. Some MELAS patients have features of the Kearns-Sayre syndrome (KSS) or myoclonic epilepsy with ragged-red fibers (MERRF), but none had the full KSS syndrome. In partial or confusing cases, analysis of mitochondrial DNA (mtDNA) may point to the correct diagnosis; however, not all patients with clinical MELAS have had the typical mtDNA point mutation and some patients with the mutation have clinical syndromes other than MELAS.


Gene therapy: present situation and future prospects.

In this review, some of the most characteristic experimental approaches have been surveyed, using in vitro cultivated cell systems or animal models to achieve therapeutical gene treatment of severe monogenic diseases. Great advances have been made in the utilization of viral-mediated gene transfer as well as by direct DNA injection techniques to permit either stable insertion of a "correcting" gene into the chromosomes of a cell or a tissue or its penetration and activity as an episome. In most instances, long-term expression of the newly introduced genetic information could be obtained and certain genetic defects could be compensated for or corrected in cultivated cells or in vivo. Recent approvals on certain protocols concerning somatic gene therapy in humans indicate that the medical world is quite conscious of the importance of this new strategy. Certain ethical considerations are listed and discussed.


Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues.

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.


Dystrophin at the plasma membrane of human muscle fibers shows a costameric localization.

We studied the distribution of dystrophin at the sarcolemma of normal human muscle fibers using high resolution immunofluorescence and confocal laser scanning optical microscopy (CLSOM). We found that the dystrophin lattice is organized at the muscle plasma membrane in an array of thick bands interconnected by a finer network. The bands encircle the muscle fiber perpendicular to the long axis of the fiber and they matched the sites of attachment of the sarcomeres to the plasma membrane. Dystrophin co-localized with vinculin, and dystrophin and vinculin co-localized with alpha-actinin at the region of the I-band. Dystrophin may be one of the proteins involved in the linkage of the sarcomeres to the extracellular matrix.


Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.


Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.


Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

A cDNA clone that encodes oryzacystatin, a cysteine protease inhibitor from rice, was isolated and expressed in Escherichia coli BL-21 (DE3) using an expression plasmid under the control of a T7 RNA polymerase promoter. The construct pT7OC 9b encoded a fusion protein containing 11 amino acid residues of the NH2 terminus of the bacterial protein phi 10 and 79 residues of oryzacystatin lacking 23 NH2-terminal residues of the wild-type protein. Recombinant oryzacystatin (ROC) constituted approximately 10% of the total bacterial protein mass and was purified in a single step by anion-exchange chromatography. The inhibitory activity of ROC toward papain (Ki = 3 x 10(-8) M) was comparable with that of the naturally occurring protein isolated from rice. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited from 18 to 85% by ROC, whereas the same activity was inhibited from 14 to 69% by the serine proteinase inhibitor phenylmethylsulfonyl fluoride. Midguts of stored product insects apparently contain both cysteine proteinases and serine proteinases, but the relative amounts vary with the species. When fed to the red flour beetle, Tribolium castaneum, 10 wt% ROC in the diet suppressed growth approximately 35% relative to that of the control group of insects.


A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.

Expression of recombinant proteins in bacteria has facilitated the characterization of many gene products. However, the biochemical characterization of recombinant proteins is limited since the bacterially expressed proteins are often synthesized as fusion polypeptides. The presence of bacterial sequences in fusion proteins further limits the use of these proteins for generating antibodies since the bacterial sequences are also antigenic. We describe two new bacterial expression vectors based on the pATH series of plasmids. These vectors were made by precisely deleting all of the trpE coding sequences found in pATH. The new vectors have enabled us to express eukaryotic genes as nonfusion polypeptides. These altered plasmids can be used to insert any DNA sequence of interest through a multiple cloning site located just 3' of an ATG start codon. Protein expression is still under the control of the trp operon and is carried out at great efficiency when the bacteria are tryptophan deprived. Studies presented here test the expression system with neurofilament subunits, NF-L and NF-H. Large amounts of recombinant nonfusion proteins were produced. Also, a time course of induction shows that the production of the nonfusion proteins was under the control of the trp operon which is readily inducible after tryptophan starvation and addition of indoleacrylic acid. These vectors may be useful for the overexpression of many proteins in a form closely approximating their native state.


Overproduction and purification of Bacillus subtilis DNA polymerase III.

The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.


Purification of the medium-chain/long-chain (COT/CPT) carnitine acyltransferase of rat liver microsomes.

A procedure for the purification of the rat liver microsomal carnitine octanoyltransferase (COT) that catalyzes the reversible formation of medium-chain and long-chain acylcarnitines from acyl-coenzyme A is described. The K0.5 for L-carnitine is 0.6 mM and the K0.5 for both decanoyl-CoA and palmitoyl-CoA is 0.6 microM. The Vmax with decanoyl-CoA is approximately fourfold greater than the Vmax with palmitoyl-CoA. The enzyme is monomeric, sodium dodecyl sulfate-polyacrylamide gel electrophoresis gives a molecular weight of 50,100, and molecular sieving gives a molecular weight of 54,300. Purified COT does not cross-react with either antimitochondrial carnitine palmitoyltransferase or antiperoxisomal COT antibodies. It also does not form a covalent adduct when incubated with etomoxiryl-CoA. Microsomal COT is a different protein than either mitochondrial carnitine palmitoyltransferase or peroxisomal COT.


The adenovirus E1A 243R protein purified from Escherichia coli under nondenaturing conditions is found in association with dnaK.

The adenovirus E1A 243R protein immortalizes primary cells in culture and induces part of the phenotypes required for transformation. It has also been shown to interact with a number of cellular polypeptides, including the product of the retinoblastoma gene. To understand more fully the molecular activities of the E1A 243R protein in association with these proteins as well as its role in the processes of cellular growth, we have developed a method for rapidly purifying this protein from genetically engineered Escherichia coli under nondenaturing conditions. The plasmid-encoded E1A protein, when expressed in a protease-deficient mutant, is found to have the same length and amino acid sequence as that which is produced in a mammalian cell. The procedure for purifying the E1A 243R protein from bacteria relies primarily upon immunoaffinity chromatography and the use of a peptide comprising the epitope recognized by an E1A-specific antibody. Elution of the E1A protein under this condition allows for gentle isolation and a purity that ranges from 90 to 96%. However, without the addition of micromolar amounts of ATP prior to its elution from the antibody column, the E1A protein is found in association with an E. coli protein of 70 kDa. Immunoblot analysis with a specific antibody showed that this bacterial protein was the heat shock protein dnaK, which is known to have extensive homology with the hsp-hsc70 family of proteins in mammalian cells. Recognition of E1A by the dnaK protein may very well reflect a situation that also occurs between the mammalian heat shock proteins and the E1A 243R protein after adenovirus infection.


Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.

A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.


A modified vector for the controlled high-level overproduction of staphylococcal protein A fusion proteins in the periplasm of Escherichia coli.

A vector encoding the Staphylococcal protein A was modified by cloning the spa gene, including its signal peptide-encoding sequence, downstream of the translation initiation sites of the phage lambda cro gene and under the control of the temperature-inducible phage lambda pR promoter. The expression from this construct was studied using the Escherichia coli phoA gene as a reporter gene after fusion to the spa gene. Determination of alkaline phosphatase activity, 1 h after temperature induction of expression at 42 degrees C, revealed an 800-fold increase over host strain background level. The presence of the alternating selectable markers on the described vector, pHEMa153, which are essential for efficient oligonucleotide-directed construction of mutations by the gapped duplex DNA method, allows the construction of recombinant and mutated forms of Staphylococcal protein A fusion proteins and efficient expression of spa gene fusions without changing the vector system.


Purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected Sf9 cells.

A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein.


A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.


Purification of an active monomeric recombinant HIV-1 trans-activator.

A method for the purification of a truncated, biologically active human immunodeficiency virus type 1 (HIV-1) trans-activator (rTAT) from recombinant Escherichia coli is reported here. The purification steps utilized include mild extraction (French press), concentration by ammonium sulfate precipitation, chromatography in 8 M urea on an S-Sepharose fast-protein liquid chromatography column, and finally, resolution by C-4 reverse-phase high-performance liquid chromatography. After the final step, the rTAT is dried and stored under salt-free conditions. Amino acid compositional analysis and N-terminal sequence analysis confirm that the purified protein is rTAT. Unlike other methods reported for purification of recombinant HIV-1 trans-activator, our protocol uses urea instead of guanidine HCl. The rTAT is fully soluble in buffered solutions at concentrations exceeding 10 mg/ml, migrates as a single 14 kDa species on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and two-dimensional PAGE gels with a pI of 9.3 +/- 0.3. Additionally, the rTAT migrates as a monomer on size-exclusion chromatography columns under native conditions. Finally, purified rTAT exhibits trans-activator activity when introduced into appropriate reporter cells. Since rTAT is monomeric when tested by gel filtration, and yet exhibits biological activity, we conclude that the method of purification we have utilized is distinct from all other methods reported to date.


High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.