The oligomeric structure of rat liver microsomal glutathione transferase studied by chemical cross-linking. The oligomeric structure of rat liver microsomal glutathione transferase was investigated using four different chemical cross-linking reagents. Studies were performed with the isolated enzyme, with the enzyme incorporated into phosphatidyl choline liposomes and with rat liver microsomes. Cross-linking was analyzed by use of SDS-PAGE combined with Western blotting. Our results strongly suggest that the microsomal glutathione transferase is a trimer in situ in the endoplasmic reticulum, as well as in the purified state and in proteoliposomes. These results lend strong support to previous studies, involving hydrodynamic characterization and radiation inactivation, indicating that the microsomal glutathione transferase is a trimeric enzyme.


Intermolecular disulfide bonds link specific high-molecular-weight glutenin subunits in wheat endosperm. A detergent wash extracted soluble proteins from wheat flour, leaving a residue enriched with insoluble glutenin aggregates. Digestion of this residue with endoproteinase Lys-C, which showed a limited specificity for glutenin subunits, produced several peptides with apparent molecular weights close to those of intact high-molecular-weight glutenin subunits. N-terminal sequencing indicated that the isolated peptides were composed of high-molecular-weight glutenin subunit fragments joined by an intermolecular disulfide bond. In two of these peptides, only two components were found, one from an x-type subunit and the other from a y-type subunit. The isolated peptides all contained at least one x-type C-terminal region and one y-type N-terminal region, suggesting a specific orientation to the intermolecular disulfide linkage.


Heterogeneous flexibilities of the active site domains of homodimeric creatine kinase: effect of substrate. (1) A single subunit and both subunits of creatine kinase from rabbit muscle was derivatized at the active site with the thiol-specific reagent 2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid. (2) The highly biphasic kinetics of the labelling reaction were characterized from measurements of activity, steady-state fluorescence and anisotropy. Derivatization of one thiol and both thiols resulted in 48 and 100% inhibition, respectively. The dead-end complex (DEC), consisting of creatine, MgADP and protein, inhibited the rate, but not the extent, of derivatization and resulted in a 2-fold increase in fluorescence. (3) The fluorescence of singlylabelled (1AANS/CK) and doublylabelled (2AANS/CK) protein exhibited three discrete lifetime components or a two-term Lorentzian distribution. The decay laws for both preparations were not remarkably different, except that, unlike 1AANS/CK, the longer decay component of 2AANS/CK was distributed, which narrowed in the presence of the DEC. (4) The steady-state anisotropies of 1AANS/CK and 2AANS/CK at 25 degrees C were 0.305 and 0.240, respectively. It was concluded that the fast reacting site was immobile and the slow reacting site was flexible. Kinetics of labelling and anisotropy emission spectra indicated that the DEC immobilized the flexible site. (5) The anisotropy decay of 1AANS/CK with and without the DEC was described by a rotational correlation time of about 50 ns, characteristic of the molecular rotation of the CK dimer. At least two terms were required to fit the data for 2AANS/CK, indicating additional segmental motion which was eliminated upon formation of the DEC. (6) Energy transfer from tryptophans to AANS indicated movement of approx. 3 A accompanying formation of the DEC.


Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator. The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)


The lytic enzyme of the Pseudomonas phage phi 6. Purification and biochemical characterization. The lytic enzyme of the lipid-containing bacteriophage phi 6, protein P5, has been purified to apparent homogeneity from disrupted viral particles. The enzyme is a monomer with a molecular mass of approx. 24 kDa. The optimal pH for P5 activity is 8.5 and the protein is readily inactivated at temperatures above 20 degrees C. Protein P5 is active against several Gram-negative bacteria, but no activity against Gram-positive species was detected. Analysis of cell wall digests indicates that P5 is not a glycosidase, but an endopeptidase splitting the peptide bridge formed by meso-diaminopimelic acid and D-alanine.


A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of a flocculent Kluyveromyces marxianus. A protein with an apparent molecular weight of 37,000 (p37) is present in very large amounts in the cell wall of Kluyveromyces marxianus, after the induction of flocculation of the yeast. This protein was isolated by preparative gel electrophoresis and its purity checked by SDS-PAGE and reverse-phase HPLC. SDS-PAGE, endoglycosidase-H treatment and peptide sequencing indicated that p37 is a glycoprotein with a high identity to cytosolic glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Polyclonal antibodies were used for Western blot analysis and immunocytochemistry, which showed that p37 is present in the cell wall of non-flocculent K. marxianus and, therefore, is a constitutive protein of the cell wall.


Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy. Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.


The predicted proteinase furin is not the hepatic proalbumin convertase. Rat and chicken liver microsomal membranes were used to investigate the relationship between proalbumin processing activity and the predicted proteinase furin. Two polyclonal antisera directed against the predicted catalytic domain of furin showed the highest level of immunoreactivity in a microsomal fraction that had minimal proalbumin converting activity. Extracts of the fraction containing most converting activity lacked detectable furin. In addition, the proalbumin convertase was not inhibited by the anti-furin antisera. These results strongly suggest that furin is not responsible for the in vivo cleavage of proalbumin.


Super helix formation of actin filaments in an in vitro motile system. Muscle contraction results from relative sliding of actin and myosin filaments. However, the possibility that actin filaments twist or rotate during sliding has not yet been experimentally investigated. We found that a super helix of an actin filament is formed in an in vitro motile system. This fact suggests that an actin filament twists and rotates due to a torque component of a sliding force generated at cross-bridges.


In vivo glycation of bovine lens crystallins. To investigate the possible role of glycation in the aging of lens proteins, we used bovine lenses as a model. We studied crystallins isolated from prenatal bovine lenses, calf lenses and lenses from mature animals (up to 20 years old). The experiments show an increase in glycation levels with age in all crystallin fractions. Regarding the lysine content of the different crystallins, gamma-crystallin showed relatively high levels of early glycation products. The results also revealed high levels of early glycation products for the HM material (containing mainly alpha-crystallin). In alpha-crystallin, alpha A-subunits were glycated to a higher extent compared with the alpha B-subunits. There is an age-related increase in advanced glycation products, measured as specific fluorescence (excitation/emission wavelengths 370/440 nm), mainly present in the HM and alpha-crystallin fraction.


Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule. The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.


Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase. Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.


Thermodynamic analysis of heparin binding to human antithrombin. The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.


Calcium dependent conformational changes of surfactant protein A (SP-A) and its collagenase resistant fragment with or without dithiothreitol. Calcium-dependent conformational changes of surfactant protein A (SP-A) and the collagenase resistant fragment (CRF) of SP-A were studied by measuring fluorescence spectra. The emission peaks of both SP-A and CRF in the absence of Ca2+ appeared at 343 nm when they were excited at 280 nm. In the presence of Ca2+, the peaks appeared at 340 nm and were accompanied by an increase in the fluorescence intensity. The magnitude of the fluorescence intensity change induced by Ca2+ was amplified by the addition of dithiothreitol (DTT) in both SP-A and CRF. The Ca2+ binding of CRF was measured by a flow dialysis method with 45CaCl2 in the Ca2+ concentration range where the Ca(2+)-induced fluorescence changes occurred. The maximum binding number of Ca2+ to CRF was about 2 mol per mol of CRF, and the value was independent of the presence of DTT.


The compact domain conformation of human Glu-plasminogen in solution. A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.


Purification and characterization of a low M(r) GTP-binding protein, ram p25, expressed by baculovirus expression system. The ram gene was isolated from rat megakaryocyte cDNA library with an oligonucleotide probe which is specific for a low M(r) GTP-binding proteins c25KG purified from human platelets. Its gene product (ram p25) is a monomeric 25-kDa guanine nucleotide-binding protein. The protein was expressed by using baculovirus transfer vector, pAcYM1, which allowed the production at a high level of soluble recombinant ram p25 in Spodoptera frugiperda (Sf9) cells under the control of polyhedrin promoter. The expressed protein in cytosol of Sf9 cells was purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. The purified ram p25 bound approx. 0.8 +/- 0.02 mol of guanosine 5'-O-1-thiotriphosphate (GTP gamma S)/mol of protein with a Kd value of 340 +/- 4.91 nM in a reaction mixture containing 10 microM of free magnesium ions. In the presence of 5 mM Mg2+, [3H]GDP was dissociated from ram p25 at the rate of 0.015 +/- 0.0010 min-1 and the dissociation was greatly enhanced by addition of 250 mM (NH4)2SO4. The rate of [gamma-32P]GTP-hydrolysis for ram p25 was 0.010 +/- 0.0012 min-1. Thus, it was indicated that the GTP-hydrolysis reaction is a rate-limiting step in the guanine nucleotide turnover of ram p25. ram p25 shares 23 and 80% amino-acid homology with the Ha-ras p21 and c25KG protein, respectively, and is similar to them in GTP gamma S binding activity in a time- and dose-dependent manner. But it differs from ras p21 in the rate-limiting step of the guanine nucleotide turnover.


Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates. Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.


Caution: the glycylmethyl and glycylethyl esters of glutathione are substrates for glyoxalase I. The glycylmethyl and glycylethyl esters of glutathione have been synthesized and carefully characterized by both 1H-NMR and tandem FAB mass spectrometry. Contrary to previously published studies, these compounds (as their methylglyoxal-thiohemiacetals) do indeed serve as moderately efficient substrates for yeast glyoxalase I, with kcat values that are approx. 3-fold smaller and Km values that are approx. 3-fold larger than those of the thiohemiacetal formed from glutathione. Product inhibition studies show that the glycylmethyl and glycylethyl esters of (S)-D-lactoylglutathione bind approx. 1.4-fold less tightly to the active site than (S)-D-lactoylglutathione. These observations exclude an essential role for the glycyl-CO2- of substrate in active site binding and catalysis.


Mechanism of ligand-protein interaction in plant seed thiamin-binding proteins. Probing the binding site of protein isolated from buckwheat seeds with a series of thiamin-related compounds. Affinities of 14 thiamin derivatives or antagonists to a thiamin-binding protein isolated from buckwheat seeds were determined. A competitive displacement of radiolabeled thiamin by unlabeled ligand was analysed by a computerized model-fitting procedure. The dissociation constant of the thiamin-protein complex was 0.93 microM. Most modifications in ligand chemical structure weakened the ligand-protein interaction. A model of the thiamin-binding site is suggested. The hydroxyethyl-chain of thiamin while protein-bound appears to be excluded from the binding region. A positively charged quaternary nitrogen atom of the thiazolium ring probably interacts with some negative group(s) of protein. The rest of the thiazolium ring as well as the amino group of the pyrimidine fragment serve as additional anchors. The three structural features of the thiamin molecule accounting for binding contribute equally to overall binding energy by about 11-12 kJ/mol.


Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli. In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.


Pressure-induced conformational changes in an antigen and an antibody and the implications on their use for hyperbaric immunoadsorption. Pressure-induced conformational changes in two proteins, bovine serum albumin (BSA) and immunoglobulin G (IgG), were studied to assess the application of hyperbaric manipulation to the dissociation of antigen-antibody complexes. Antigen-antibody dissociation is important in the product-recovery phase of immunoadsorption, an affinity purification process. Three techniques were used in parallel for this study, including fluorescence, Fourier transform infrared (FTIR) spectroscopy, and the enzyme-linked immunosorbent assay (ELISA). Employing a fluorescent probe, fluorescent intensity measurements were used to detect protein conformational changes. FTIR spectroscopy was used to determine changes in protein secondary structure induced by high pressure, while the ELISA test was used to examine antibody recognition after the proteins had been pressure-treated. The results from this work demonstrate that IgG is resistant to conformational changes induced by pressures below 2 kbar. In contrast, BSA undergoes reversible conformational changes in this pressure range. However, these conformational changes are not reflected in tests measuring antibody recognition. These findings indicate that IgGs have the potential to be used as recycled ligands in immunoadsorption separation processes. Different antigens that are being considered for purification by immunoadsorption and separated by means of high pressure could be screened by the methods disclosed to determine their stability under high pressure conditions.


FTIR spectroscopic kinetic analysis of alkaline phosphatase under hyperbaric manipulation. For the first time, high-pressure infrared spectroscopy has been used in an enzyme kinetics study. This technique allows not only the investigation of kinetics under very high pressure, but it also allows simultaneous determinion of changes in the secondary structure of enzymes at the corresponding pressures. In the present study, a classical enzyme reaction, the conversion of p-nitrophenol phosphate into p-nitrophenol by alkaline phosphatase was selected to demonstrate the potential of infrared spectroscopy as an alternative physical method in the high-pressure study of enzyme kinetics. The rate constants of this enzyme reaction have been determined as a function of pressure in the pressure range 0.001-14 kbar. The first-order rate constants thus obtained increases with increasing pressure up to 8.3 kbar. At this pressure, the reaction rate decreases abruptly due to the denaturation of the enzyme arising from the conformational changes of some alpha-helical segments in the enzyme molecules into beta-sheet structure. The present results suggest that the pressure-enhanced overall hydrogen-bond strength in the amide groups of the enzyme is one of the factors which stimulate the enzyme activity. Moreover, the dissociation of the dimeric enzyme into its subunits does not inhibit the enzyme activity but only attributes to a slight change in activation volume.


Components and proteolytic processing sites of arylsulfatase B from human placenta. Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.


Chemical modification of arginine residues in alpha-bungarotoxin. The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.


Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue. Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.


Nucleotide- and temperature-induced changes in myosin subfragment-1 structure. The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.


Lactoperoxidase-catalyzed oxidation of thiocyanate ion: a carbon-13 nuclear magnetic resonance study of the oxidation products. Products formed from the lactoperoxidase (LPO) catalyzed oxidation of thiocyanate ion (SCN-) with hydrogen peroxide (H2O2) have been studied by 13C-NMR at pH 6 and pH 7. Ultimate formation of hypothiocyanite ion (OSCN-) as the major product correlates well with the known optical studies. The oxidation rate of SCN- appears to be greater at pH < or = 6.0. At [H2O2]/[SCN-] ratios of < or = 0.5, OSCN- is not formed immediately, but an unidentified intermediate is produced. At [H2O2]/[SCN-] > 0.5, SCN- appears to be directly oxidized to OSCN-. Once formed, OSCN- slowly degrades over a period of days to carbon dioxide (CO2), bicarbonate ion (HCO3-), and hydrogen cyanide (HCN). An additional, previously unrecognized product also appears after formation of OSCN-. On the basis of carbon-13 chemical shift information this new species is suggested to result from rearrangement of OSCN- to yield the thiooxime isomer, SCNO- or SCNOH.


Long range electron transfer along an alpha-helix. The many observations of long range electron transfer in proteins raises the question of whether a protein's structure can influence the rate or path of such transfers, and if so, then how. To answer these questions requires information on which of the various structural elements composing proteins support long range electron transfer. In this report, we present evidence for long range electron transfer along the alpha-helix of a synthetic leucine zipper dimer. We also present electron transfer rate data obtained with other helical peptides.


Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations. Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.


Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin. The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.


Clusterin binds by a multivalent mechanism to the Fc and Fab regions of IgG. Clusterin was purified from human serum by IgG and monoclonal antibody affinity chromatography. SDS-PAGE and immunoblotting revealed no major differences between clusterin prepared in these two ways. An ELISA method for measuring the binding of clusterin to immunoglobulins was developed. Clusterin purified by IgG affinity chromatography bound to pooled human IgG with a similar affinity (S0.5 5.9 +/- 0.4 micrograms/ml) as clusterin purified by monoclonal antibody chromatography (S0.5 6.1 +/- 0.2 micrograms/ml). The apparent affinity of clusterin for IgG immobilised on ELISA plates increased with increasing concentrations of IgG in the coating solution. Aggregated IgG in solution was a more potent inhibitor of the binding of clusterin to immobilised IgG than was monomer IgG. Clusterin bound to all of the isotypes of human IgG, and to human IgA and IgM, with apparent affinities in the order IgG3 > IgG4 > IgM > IgG1 > IgG2, IgA. Clusterin bound to both the Fab and Fc fragments of human IgG. The clusterin binding site(s) on the Fc do not overlap with those for protein A and Clq.


Albumin Rugby Park: a truncated albumin variant caused by a G-->C splice-site mutation in intron 13. Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.


Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant. Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.


A new, simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS. In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method is demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.


The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies. Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.


A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene. A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3' end of the coding region. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the zeta-crystallin gene disclosed a dinucleotide deletion of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.


Hemoglobin Rouen (alpha-140 (HC2) Tyr-->His): alteration of the alpha-chain C-terminal region and moderate increase in oxygen affinity. Hb Rouen (alpha 140(HC2) Tyr-->His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the alpha-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the beta-chain. This model provides additional evidence for the inequivalence between the alpha- and beta-subunits.


Immunochemical characterisation of parathyroid hormone-related protein from tumour and non-tumour cells. The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtration chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1-86 and 1-34) and bioactivity (1-34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19-22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10-16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1-34 or 37-67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24-25 kDa, consistently slightly larger than recombinant PTHRP(1-141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.


Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metabolite secreted as the 11,12-epoxy-eicosatrienoic acid. The development of long-term culture of AIDS-KS cells has allowed us to investigate further a possible vascular origin of Kaposi sarcoma. Taking into account the relative specificity of arachidonic acid (AA) metabolism according to cell type, the AA 'cascade' was analyzed in cultured KS-3 cells established from lung biopsies and compared to human umbilical venous endothelial (H-UVE) cells and human myometrial smooth muscle (H-MSM) cells, under basal conditions and after stimulation with vasoactive agents such as histamine or thrombin. Considering strictly the 'prostaglandin' profile given by RIAs, the metabolism of AA was closer, whilst not identical, to H-UVE than to H-MSM cells. However, evaluation of all the eicosanoids released from [3H]AA labeled KS-3 cells revealed that the predominant metabolite was not prostacyclin (PGI2), as suggested from PG RIAs, but an epoxy-eicosatrienoic acid (EET), identified as the 11, 12 isomer by HPLC and MS/MS. The synthesis of this EET is probably cytochrome P-450 monooxygenase dependent. Its potential role in the development of the KS tumor cells is under investigation.


Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist. We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothioglucose, which has disease-modifying activity in treated patients, acts as an IL-1 functional antagonist in this system.


Characterization of two HEXB gene mutations in Argentinean patients with Sandhoff disease. Beta-hexosaminidase A (beta-N-acetyl-D-hexosaminidase, EC 3.2.1.5.2) is a lysosomal hydrolase composed of an alpha- and a beta-subunit. It is responsible for the degradation of GM2 ganglioside. Mutations in the HEXB gene encoded beta-subunit cause a form of GM2 gangliosidosis known as Sandhoff disease. Although this is a rare disease in the general population, several geographically isolated groups have a high carrier frequency. Most notably, a 1 in 16-29 carrier frequency has been reported for an Argentinean population living in an area contained within a 375-km radius from Córdoba. Analysis of the genomic DNA of two patients from this region revealed that one was homozygous for a G to A substitution at the 5' donor splice site of intron 2. This mutation completely abolishes normal mRNA splicing. The other patient was a compared of the intron 2 G-->A substitution and a second allele due to a 4-bp deletion in exon 7. The beta-subunit mRNA of this allele is unstable, presumably as a result of an early stop codon introduced by the deletion. Two novel PCR-based assays were developed to detect these mutations. We suggest that one of these assays could be modified and used as a rapid screening procedure for 5' donor splice site defects in other genes. These results provide a further example of the genetic heterogeneity that can exist even in a small geographically isolated population.


Electrophysiological follow-up of acute and chronic experimental allergic encephalomyelitis in the Lewis rat. Cortical somatosensory evoked potentials (c-SEP) and flash visual evoked potentials (f-VEP) were serially recorded in acute monophasic and chronic relapsing experimental allergic encephalomyelitis (EAE) in the Lewis rat. In acute EAE, a significantly delayed latency and broadened peak of the c-SEP were observed corresponding to the clinical onset, and then returned to normal with the disappearance of clinical signs. In chronic EAE, the c-SEP showed the same changes as in acute EAE, also reflecting the first attack, remission and relapsing phase. However, chronic EAE, when paralysis had recovered in the relapsing phase, showed c-SEP abnormalities suggestive of subclinical active lesions. In contrast, the f-VEP showed no obvious abnormalities in acute or chronic EAE. These findings suggest that the c-SEP is an objective and sensitive index for detecting clinical and pathological changes in acute and chronic EAE in the Lewis rat.


Prospective long-term follow-up of depressed patients with and without suicide attempts. This 4-6-year prospective follow-up study compared reactive depressives with (n = 48) and without suicide attempts before index admission (n = 24). Both groups showed a favourable course and outcome concerning psychiatric diagnoses (DSM-III), psychopathology, social integration, and social functioning as well as displaying a nearly identical course and outcome. In both groups, two patients committed suicide attempts during the follow-up period. 2 (1) male patients from the group with suicide attempts committed suicide.


Recurrent brief depression and its relationship to seasonal affective disorder. Recurrent brief depression (RBD) and seasonal affective disorder (SAD) have been both recently described as subgroups of major depression (DSM-III-R). We have established a relationship between these two syndromes in a cohort of 42 outpatients who presented themselves to a clinic for seasonal affective disorder at the Psychiatric Department of the University of Bonn, FRG. Our preliminary data indicate that 31% of the patients who were diagnosed as suffering from either SAD or its subsyndromal form (S-SAD) can also be categorized as RBD (RBD-seasonal) in a 1-year observation period. During the time span of 1 year RBD-seasonal patients had a mean number of 20 (SD 9) episodes compared with 6 (SD 5) episodes (P less than 0.001) in the group of seasonal patients without BRD. These episodes were accentuated in fall/winter and outnumbered those in spring/summer significantly (P less than 0.001). The mean duration of each episode was 4.6 (SD 2.6) days in the RBD-seasonal group and 21.8 (SD 29) in the non-RBD-seasonal group. Patients with RBD-seasonal experienced seasonal changes as more of a problem and reported a lower percentage of first-degree relatives with a history of depression than the non-RBD-seasonal group.


Schizoaffective disorders with and without onset in the puerperium. The premorbid and sociodemographic features, long-term course and long-term outcome (on average 23.8 resp. 26.8 years after onset of illness) were compared in 30 female schizoaffective patients with onset of their illness during the puerperium and 60 female schizoaffective patients with onset at other times. The majority of premorbid and sociodemographic variables as well as course parameters were similar in the two groups. Most of the few differences (in age at first manifestation, marital state at onset, presence of stable heterosexual relationship before onset, acuteness of onset, presence of life events) are closely connected with the inclusion and exclusion criteria applied for the puerperal disorders (exclusion of patients with preexisting illness or psychiatric symptoms during pregnancy, inclusion only if onset was within 6 weeks of parturition). The puerperal schizoaffective disorders began more frequently with a schizomanic episode and less frequently with a schizodepressive episode than did the non-puerperal schizoaffective disorders, a finding which perhaps reflected the "pathoplastic" role of the puerperium on psychotic disorders. Several significant differences were found regarding the long-term outcome (frequency of persistent alterations, level of global functioning and disability, non-achievement of the expected social development, loss of autarky), confirming earlier findings that puerperal disorders generally have a better outcome than other psychotic disorders.


Is the DSM-III-R category of mood disorders too broad? Personality findings. In the DSM-III and DSM-III-R the affective or mood category has been widened and mood-incongruent psychotic affective illness (MICPAI) included. The present study was undertaken to determine whether this broad mood category is still homogeneous. Personality factors were used as parameters. Minnesota Multiphasic Personality Inventory findings of 54 patients with MICPAI were compared with those of 21 probands with a DSM-III typical affective disorder and with those of 15 DSM-III schizophrenics. It was shown that MICPAI differed significantly from typical affective disorder, but not from schizophrenia, in particular regarding the subscales "schizophrenia" and "psychopathic deviate". When MICPAI was subdivided into the depressed and manic type, the depressed type was found to be more closely related to schizophrenia (with respect to the subscales "paranoia" and "schizophrenia"), whereas the manic type hardly differed from affective disorder. Whether this result is due to diagnostic inaccuracies is discussed. Our finding that MICPAI differs from typical affective disorder with respect to personality is in accordance with heredity and outcome studies demonstrating that MICPAI is associated with a higher risk for schizophrenia in first-degree relatives and with worse outcomes when compared with typical affective disorder. It can thus be concluded that the decision to include MICPAI in the affective or mood category of the DSM-III or DSM-III-R has rendered this category more heterogeneous.


Functional motor asymmetries correlated with clinical findings in unmedicated schizophrenic patients. Motor asymmetries were investigated in 28 unmedicated schizophrenic in-patients and 32 healthy controls. Of the patients, who were assessed as right-handers by a handedness questionnaire, 46.4% changed their motor laterality in at least one part of the tapping test series, probably because of a decrease of functional hemispheric asymmetry. These patients were characterized by more pronounced psychotic symptoms than those who did not change motor laterality. According to the tapping frequency, two groups of patients could be distinguished: the high- and the low-frequency group. In both groups certain tapping data could be correlated with characteristic clinical features. While the findings in the high-frequency group point to an enhanced activation level of the right hemisphere and appear to be correlated with the onset of positive symptoms and better prognosis, the findings in the low-frequency group may be a reflection of a disturbed function of the left frontal region and seem to be correlated with a gradual chronic development of predominant negative symptoms with worse prognostic implications.


Facial expression and emotional face recognition in schizophrenia and depression. Twenty-three acute schizophrenics, 21 acute major depressives (Research Diagnostic Criteria), and 15 normal controls participated in a study on facial expression and emotional face recognition. Under clinical conditions, spontaneous facial expression was assessed according to the affective flattening section of the Scale for the Assessment of Negative Symptoms. Under experimental laboratory conditions involuntary (emotion-eliciting interview) and voluntary facial expression (imitation and simulation of six basic emotions) were recorded on videotape, from which a raterbased analysis of intensity or correctness of facial activity was obtained. Emotional face recognition was also assessed under experimental conditions using the same stimulus material. All subjects were assessed twice (within 4 weeks), controlling for change of the psychopathological status in the patient groups. In schizophrenics, neuroleptic drug influence was controlled by random allocation to treatment with either haloperidol or perazine. The main findings were that schizophrenics and depressives are characterized by different quantitative, qualitative, and temporal patterns of affect-related dysfunctions. In particular, schizophrenics demonstrated a trait-like deficit in affect recognition and in their spontaneous and voluntary facial activity, irrespective of medication, drug type and dosage, or extrapyramidal side-effects. In depressives a stable deficit could be demonstrated only in their involuntary expression under emotion-eliciting interview conditions, whereas in the postacute phase a reduction in their voluntary expression became apparent. Differences in patterns of affect-related behavioral deficits may reflect dysfunctions in different underlying psychobiological systems.


Nocturnal plasma levels of cytokines in healthy men. Nocturnal cytokine levels were measured serially in 12 healthy male volunteers for 12 h, including 8 h of polygraphically monitored nocturnal sleep. Plasma concentrations of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were determined in 30-min intervals by enzyme-linked immunoadsorbent assays. In some subjects cytokines were not detectable at all. In the remaining volunteers (27% for IL-1 beta, 58% for IL-6 and TNF-alpha, respectively) occasional values near to the detection limits (DL) of the assays could be measured. With respect to IL-1 beta and IL-6, plasma levels above the DL were significantly more frequent during sleep than during the preceding time of wakefulness. No temporal association with NREM or REM episodes could be shown. TNF-alpha values above the DL were randomly distributed across the 12-h period investigated. It is concluded that in a considerable percentage of healthy subjects small amounts of cytokines are released at night. Release of IL-1 beta and IL-6 is temporally associated with sleep, whereas the release of TNF-alpha is not. It remains to be established whether nocturnal cytokine release reflects either an interaction between sleep and host defense mechanisms or a sleep-independent circadian rhythmicity.


Transnational stability of gender differences in schizophrenia? An analysis based on the WHO study on determinants of outcome of severe mental disorders. Gender-specific analyses of the multinational WHO-Determinants of Outcome-Study (including 1,292 cases from 10 countries) demonstrate the transnational stability of major findings on gender differences in schizophrenia: Male patients have an earlier mean age at onset in all countries. In female patients, the distribution of the age at onset shows a second peak after age 40 years. No gender differences on nuclear symptoms of schizophrenia can be detected, but on uncharacteristic symptoms, particularly some aspects of the illness behaviour, differences appear. This investigation supports the transcultural validity of gender differences found in the German ABC-Schizophrenia-Study and in the Danish-German Psychiatric Case Register studies.


Body weight and water intake of guinea pigs: influence of single caging and an unfamiliar new room. No changes in body weight are observed if adult male guinea pigs are removed from their groups and kept singly in their home enclosure. In contrast, subjects markedly lose weight and show a marked reduction of water intake if isolated from their groups and caged singly in an unfamiliar test room. It is recommended that if guinea pigs have to be caged singly, they should remain in their home room, for enabling them to express their need for oldfactory/auditory contact with conspecifics.


Microvascular transposition of the arterial supply to the testis of the boar. The origin of the arterial supply to a testis was changed in 7 boars with a microsurgical technique. A. testicularis was connected, end to side, to a branch of the femoral artery. The capillary blood flow was unchanged, indicating viable transplants. The temperature difference between testis and body disappeared after the surgery, indicating an interruption of the heat transfer in the pampiniform plexus. The lower values of the histological score and the possibly lower peripheral plasma concentration of testosterone after the arterial by-pass may indicate a physiological importance of the counter current exchange.


Cardiac arrhythmias during experimental induction and management of myocardial infarction in the dog. The experimental production of a myocardial infarction (MI) in the dog was achieved adopting a new catheter technique. After induction of the MI, a variety of arrhythmias appeared and were classified according to the Lown-classification as more or less severe. The therapy was achieved with antiarrhythmics class 1-111 (Vaughn-Williams classification) for late premature ventricular beats or couplets. Sinus tachycardia was often terminated by occular or sinus carotis pressure or a new selective sinus blocking agent. Results showed Amiodarone to be the drug of choice in the treatment of severe arrhythmias (Lown IV and V) like triplets or salves of extrasystolies. Ventricular fibrillation always resulted in the death of the animal, because fibrillation was not convertible by direct current cardioversion, endovenous injection of Lidocaine or even internal cardiac massage. The registration of hemodynamic parameters (left ventricular end diastolic pressure, wedge pressure, pulmonary and aortic pressure) was shown to be important in controlling the therapy, as well as blood gas and electrolyte analysis.


Evaluation of a psychological treatment package for treating pain in juvenile rheumatoid arthritis. We examined the utility of psychological treatment procedures for children with high levels of pain associated with juvenile rheumatoid arthritis (JRA). By the use of a multiple baseline across subjects design, four children were assigned to an immediate treatment group, and four children to a delayed treatment group. The six-session treatment included relaxation training, electromyogram, and thermal biofeedback for the child; mothers were trained in the use of behavioral techniques for managing physical therapy and school attendance. Visual inspection of the data indicates small changes on children's self-reported pain diary scores for mean pain and ratings of high (greater than 5 on a 10-point visual analogue scale) pain periods, with 50% to 62% showing at least a 25% reduction in pain immediately after treatment, and 62% to 88% showing a 25% reduction by 6-month follow-up. Maternal reports of changes paralleled those of the children. Comparisons of Mann-Whitney U-tests conducted pre- and posttreatment indicated no differences for children's ratings of mean pain or +5 pain ratings between the immediate and delayed treatment groups; greater improvement for the immediate treatment group was noted on maternal reports of both mean pain (p < 0.05) and +5 pain (p < 0.5) ratings. The reduction of pain reports from pretreatment to follow-up was significant for children's mean pain (p = 0.02), +5 pain ratings (p = 0.02), and mother's reports of mean pain (p = 0.03) and +5 pain periods (p = 0.01). Maternal reports of the number of pain-related behaviors that the child exhibited also declined (p < 0.05). No reduction in physical therapist's ratings of pain during evaluation were noted. No increases in maternal reports of child's psychological adjustment problems were reported following treatment. Results provide modest support for the use of psychological interventions with patients with JRA.


Changing patterns of hospital use for patients with musculoskeletal disease in Michigan, 1980 to 1987. Over the past 10 years there have been dramatic changes in health care financing in the United States, such as Medicare's Prospective Payment System for hospitalized Medicare beneficiaries, and in health services delivery, such as the growth in health maintenance organizations and other forms of managed care. These changes have occurred largely in response to payors' concerns about the rising cost of health care. A study of such changes in financing and delivery, and how specific groups of patients are affected is necessary so that the effects of these changes on patients' health can be determined. We examined the hospitalization rates for patients with musculoskeletal diseases in Michigan from 1980 through 1987. During this period, the overall age-adjusted hospitalization rates decreased 7.0% per year (p = 0.001). The decrease occurred less for surgical discharges (6.0% per year) than for medical discharges (8.6% per year) (p < 0.001). While these overall trends are of interest, they obscure disease-specific trends that vary significantly from both the overall, and the medical and surgical trends. For example, while surgical discharges, in general declined, procedures related to major joint and limb reattachment (DRG #209) increased at a rate of 6.3% per year. And while medical discharges in general decreased over this period, discharges for osteomyelitis increased 5.4% per year. The patterns of disease-specific trends offers insight into the possible causes for these changes. Finally, it is important to understand the epidemiology of hospital use to evaluate the effects of new medical care delivery and payment systems on the care of subsets of patients.


Validation of the NIH activity record: a quantitative measure of life activities. This article reports the results of the validation of a life activity record. We devised a self-administered daily log (the NIH Activity Record, ACTRE), for persons with rheumatoid arthritis (RA), which recorded specific daily activities over a 24-hour period and identified the level of physical effort for each task. In addition, each activity was assigned a level of pain, fatigue, difficulty, competence, meaningfulness and enjoyment. Twenty-one persons with RA completed the log. They underwent an articular examination (AI) (Ritchie Articular Index) as well as completed the following self-reports: Psychosocial Adjustment to Illness Scale (PAIS); The Feeling Tone Checklist (FTC), a measure of fatigue; The Modified Health Assessment Questionnaire (ALI); and the Pain and Disability Index (PDI). Significant correlations were found between fatigue measured by ACTRE and FTC (p = 0.028); pain measured by ACTRE, PDI (p = 0.002), and (p = 0.01) and the visual analog scale in the ALI (p = 0.0002). Pain experienced while performing self-care measured by ACTRE correlated with AI (p = 0.001) and ALI (p = 0.0013). Difficulty with self-care activities on the ACTRE correlated with difficulty (p = 0.007) and pain (p = 0.012) on the ALI. The ACTRE is a valid measure of symptoms and perceptions that can be quantified, and is unique in that it identifies specific daily activities likely to produce them.


Effect of active hand exercise and wax bath treatment in rheumatoid arthritis patients. The effect of active hand exercise and warm wax treatment was evaluated in 52 rheumatoid arthritis patients randomized into four groups: (1) both exercise and wax bath, (2) exercise only, (3) wax bath only, and (4) controls. Treatment was given three times a week for 4 weeks. Deficits in flexion and extension in digits II-V bilaterally, grip function, grip strength, pain, and stiffness were measured before and after the treatment period. The control group was measured at corresponding times. Wax bath treatment followed by active hand exercise resulted in significant improvements of range of motion (ROM) and grip function. Active hand exercise alone reduced stiffness and pain with nonresisted motion and increased ROM. Wax bath alone had no significant effect.


Physical fitness levels in children with polyarticular juvenile rheumatoid arthritis. Children with juvenile rheumatoid arthritis (JRA) often exhibit fatigue and prolonged exercise recovery. Improved fitness through physical conditioning has not been a goal of standard medical or physical treatment regimens for JRA, and fitness levels of children with JRA have rarely been studied. We compared physical fitness in 20 6 to 11-year-old patients with polyarticular JRA with sex-, age-, and size-matched controls, using the Health Related Physical Fitness Test (HRPFT), a national, standardized, norm-referenced test. We correlated fitness scores with summary joint counts, and with an articular severity index (sum of joint swelling, tenderness, pain, and limited range for each child). The results showed that children with polyarticular JRA were less physically fit than normally active (noncompetitively athletic) children of the same sex, age, and size. There was no statistically significant relationship between increased joint counts, and/or disease severity scores, and reduced fitness scores. This suggests that physical fitness levels are less related to degree of "disease activity" than is often thought. We conclude that (1) a readily available, nationally standardized fitness test can be used to assess children with JRA: and (2) fitness levels and measures of disease activity do not correlate. We believe that multiple factors, perhaps including family, physician, and school concerns about potential disease exacerbation following exercise, may account for the low fitness levels observed in children with JRA.


Uptake by macrophages of a biotinylated oligo-alpha-deoxythymidylate by using mannosylated streptavidin. Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(alpha-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis (alpha-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.


Covalent linkage of ruthenium polypyridyl compounds to poly(L-lysine), albumins, and immunoglobulin G. A series of ruthenium polypyridyl complexes has been covalently bound to poly(L-lysine), bovine serum albumin, human serum albumin, ovalbumin, and immunoglobulin G using different binding methods. The conjugation ratios and the luminescence properties of the bioconjugates are reported. All conjugates show nonsingle-exponential decay curves. Quenching of the emission by oxygen has been studied.


Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin. We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.


Light-induced coupling of aqueous-soluble proteins to liposomes formed from carbene-generating phospholipids. A novel bilayer-forming phospholipid analogue with a photoactivatable carbene-generating head group was synthesized and characterized with respect to molecular structure and light-induced reactivity. N'-(1,2-Dimyristoyl-sn-glycero-3-phosphoethyl)-N-[m-[3- (trifluoromethyl)diazirin-3-yl]phenyl]thiourea (PED) was prepared by thiocarbamoylation of synthetic dimyristoylphosphatidylethanolamine with 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine. PED formed liposomes in aqueous media. Gel to liquid-crystalline transitions occurred at 10.5 degrees C. Neither PED- nor PED/dimyristoylphosphatidylcholine mixed liposomes underwent major structural changes when photoactivated. Liposome sizes, determined by electron microscopy, were not altered upon light exposure. PED combines the advantages of facile synthesis and timed carbene reactivity by photoactivation at wavelengths greater than or equal to 320 nm. Conditions used for PED photoactivation did not inactivate catalytically active or complex-forming proteins. Light-induced binding of aqueous-soluble proteins to PED containing liposomes was attained through photoactivation in the presence of myoglobin, streptavidin, or trypsin. The proteins mentioned were utilized to characterize carbene-initiated ligand coupling. Procedures described establish a new and versatile method for the formation of proteoliposomes.


[Inhibition of total and active E-rosettes mediated by serum from tubercular patients]. To study the effect of tuberculous patients' serum upon normal lymphocytes in order to determine possible inhibition factors of immune response. We studied total E-rosette formation (4 degrees C, RET) and active rosette formation (37 degrees C, REA) in tuberculous patients in different stages: mild (n:36), moderate (n:28), severe (n:24) and a control group of 32 patients. The same determinations were studied in normal circulating lymphocytes (LN) incubated before with serum from tuberculous patients (SP), serum of normal individuals (SN) and in Tc-199 medium. The number of RET and REA in tuberculous patients of different stages were significantly lower to the ones of the control group (p less than 0.01, p less than 0.05). This differences were also found when we compare the number of RET and REA after the incubation of LN with SP and SN respectively (p less than 0.01, p less than 0.05). Tuberculous patients showed an impaired ability to form RET and REA. The incubation of normal lymphocytes with serum from tuberculous patients reduced significantly its capacity to form rosettes.


Preparation of micelle-forming polymer-drug conjugates. Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.


[Tuberculosis in 7 general hospitals in Andalusia. Grupo Andaluz para el Estudio de las Enfermedades Infecciosas]. To know the clinical features of tuberculosis in our environment, to evaluate its diagnostic techniques and therapeutic options as well as the evolution of patients. Multicenter retrospective study of 1115 patients with tuberculosis, diagnosed between 1984 and 1988 in the population based areas of 7 Hospitals from Andalusia (Spain). The mean age was less than 40 ages, the exponential growing of the number of cases a year in which the influence of drug addicts could be an important factor, an elevated proportion (45%) of extrapulmonary tuberculosis and disseminated forms, and a social environment of 20% of cases being alcoholics or drug addicts. A good use of diagnostic techniques is recorded, although the use of culture as diagnostic tools is lacking. The usual treatment was three drugs for nine months. The global evolution seems good. However a global mortality of 6.4% is recorded, mainly in disseminated forms and among patients with risk factors for developing tuberculosis. We have seen an increment among tuberculosis cases, as well as a change in the clinical spectrum of the disease, linked to social illness and drug addiction. The diagnostic approach to tuberculosis seems to be appropriate. The follow up of patients is somewhat confusing.


[Serodiagnosis in pulmonary tuberculosis. Clinical evaluation]. The IgA, IgG, IgM and IgG subsets antibodies levels were determined in 200 patients with pulmonary tuberculosis and compared to three control groups: 80 healthy individuals (50 with negative PPD skin test, 30 with positive PPD skin test), 30 leprosy patients and 20 patients with different pulmonary diseases. The technique used was an enzyme linked assay. As antigens, purified tuberculin and Ag60 from M. bovis were used. There were not statistically significant differences between antibody levels among all control groups studied, but when we compare the level in control groups with that observed in tuberculous patients, they showed higher levels of IgA, IgG, IgM, IgG2 (p less than 0.01) and IgG4 (p less than 0.05). A definite diagnosis of tuberculosis of the lung should only be established if the patient showed to be positive to IgG plus IgA or IgM and in special cases to IgG1, reaching then a diagnostic efficacy of 90% in a patient population with a 68% of positive smears for acid-fast bacilli.


[Antigenic response against PPD and antigen 60 in tubercular patients: single antigen versus the combined test]. We analyze serum samples from 70 patients with pulmonary tuberculosis and 50 healthy individuals. The antigenic activity (IgG) against protein purified antigen (PPD) and antigen 60 (A60) from M. tuberculosis. Thirteen patients were also HIV infected, and three patients had AIDS defined by the presence of disseminated tuberculosis. The test using antigen alone showed a 77% sensitivity and 74% specificity when PPD is used. When A60 was used, both values improved (81% sensitivity, 94% specificity). The use of a combined test (PPD and A60) improves the sensitivity (89%) but reduces the specificity (82%). The HIV infected patients showed similar responses to those of other patients. The combined use of different antigens might be useful for diagnosing tuberculosis.


An improved method for labeling monoclonal antibodies with samarium-153: use of the bifunctional chelate 2-(p-isothiocyanatobenzyl)-6- methyldiethylenetriaminepentaacetic acid. Samarium-153 (153Sm) radioimmunoconjugates of the monoclonal antibody K-1-21 were produced using the bifunctional chelate 2-(p-isothiocyanatobenzyl)-6- methyldiethylenetriaminepentaacetic acid (Mx-DTPA). The specific activity (up to 150 MBq mg-1) and percent retained immunoreactivity (greater than 75%) were similar to that of 153Sm-K-1-21 conjugates formed with cyclic DTPA anhydride (cDTPAa). In vivo biodistribution studies showed specific localization of 153Sm-Mx-DTPA-K-1-21 to target antigen implants and higher blood pool and lower uptake in liver, spleen, kidney, and bone when compared to 153Sm-cDTPAa-K-1-21. The improved in vivo distribution of 153Sm-Mx-DTPA-K-1-21 should result in lower radiotoxicity to nontarget tissues when used for radioimmunotherapy purposes.


[DNA amplification using PCR in the diagnosis of tuberculosis]. To evaluate the DNA amplification technique of M. tuberculosis with PCR in clinical samples. We have studied 57 clinical samples of 49 patients using PCR and culture in conventional media. We evaluate the final diagnosis of tuberculosis depending upon the bacteriologic results of all available samples. We reached a 58% sensitivity and 100% specificity. In 9 samples with positive PCR the culture was negative. The samples belonged to 9 patients with tuberculosis (4 with active disease and 5 with past microbiological diagnosis). In 32 samples with negative PCR test, the microorganism was isolated in 9 cases. More studies are needed before PCR technique could be recommended for widespread use in the diagnosis of tuberculosis. Its long duration, the equipment needed and the false negative results are among its limitations.


Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells. A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.


[Isolation of Yersinia enterocolitica 0:8 in Spain]. We presented three cases of enterocolitis due to Yersinia enterocolitica 0:8. We use common isolation media, pathogenicity markers and plasmid analysis. The study of genetic and biochemical characteristics of the strains showed that they belonged to 1B biotype, 0:8 serotype, and that they not carry the virulence plasmid. However, they have pathogenicity markers that were considered typical of highly virulent strains. This are the first isolations of this particular serotype in Spain and they showed, as it happens in other European countries, the spread of serotype 0:8 around the world.


[Osteoarticular infections in children]. We presented a retrospective review of osteoarticular infections diagnosed at Sant Joan de Deu Hospital from Barcelona, in the last 10 years (1981-1990). A total of 127 arthritis and 113 osteomyelitis were recorded in children aged from a few days to 15 years. This represents a 3.5% of all infectious diseases patients during the study period. The most common microorganism was S. aureus (52.3%) followed by Brucella (8.2%) and Salmonella as well as Streptococcus agalactiae (7.3% each). The most common involved joint were hip and knee in arthritis cases, but long bones (tibia and humerus) in osteomyelitis cases.


Photoaffinity labeling of scallop myosin with 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate: identification of an active site arginine analogous to tryptophan-130 in skeletal muscle myosin. The ADP photoaffinity analogue 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP) was used to photolabel the ATP binding site of scallop myosin. Approximately 1 mol of NANDP per mol of myosin was trapped at the active site by complexation with vanadate and manganese. ADP, but not AMP, inhibited trapping of NANDP. The trapped NANDP photolabeled up to 37% of the myosin upon UV irradiation. Papain subfragment-1 prepared from the photolabeled myosin was digested with trypsin, and the major photolabeled tryptic peptides were isolated by reversed-phase HPLC. The amino acid sequence of the major labeled peptide was X-Leu-Pro-Ile-Tyr-Thr-Asp-Ser-Val-Ile-Ala-Lys, where X represents the photolabeled amino acid Arg128. Previously, Trp130 of rabbit skeletal muscle myosin has been shown to be photolabeled by NANDP [Okamoto, Y., and Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1580]. Scallop and rabbit skeletal muscle myosin display a high degree of sequence similarity in this region with Arg128 in an equivalent position as Trp130. These results suggest that the composition of the purine binding site is analogous in both myosins and that Arg and Trp play a similar role in binding ATP, despite the marked differences of their side chains.


[Comparison of the new Granada medium and the ICON-strep B test in the detection of group B streptococci in pregnant patients]. The detection of group B streptococci by the ICON Strep-B test an enzyme immunoassay, and culture in new Granada Medium was compared in tubes and plates in a total of 200 vaginal specimens from pregnant women. The group B streptococci were cultured in new Granada medium from 33 of these specimens (incidence of 16.5%). Compared with culture in new Granada medium, the sensitivity and specificity of the ICON-Strep B test were 66.6 and 100%, respectively. Because of their poor sensitivity, we do not recommend the use of these rapid test as the only basis for GBS detection.


Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. A simple spectrophotometric method was developed to quantitate micromolar concentrations of a bifunctional DTPA ligand in DTPA monoclonal antibody (mAb) conjugates. Titration of a brightly colored 1:2 yttrium (III) complex of arsenazo III with the ligand 1B4M-DTPA obeyed Beer's law over the concentration range 0-2.0 microM 1B4M-DTPA at 652 nm. From a calibration plot of absorbance versus 1B4M molarity, concentrations of 1B4M-DTPA conjugated to mAb were determined. Mole ratios of 1B4M-DTPA to mAb agreed satisfactorily with the ratios obtained by a radioanalytical technique using carbon-14-labeled 1B4M-DTPA and a binding assay using 111In. The spectrophotometric method was applied successfully to the preparation of 1B4M-DTPA mAb anti-TAC, a mAb conjugate used in clinical trials of 90Y radioimmunotherapy.


[Automated method for yeast identification: ATB 32 C]. The aim of this study was to evaluate the ATB 32 C (API system) automatic medium for identifying yeasts in clinical samples. A total of 101 yeasts strains were studied, representing 8 genera and 18 different species, identified by conventional means. All 32 microdomes of the track, including dehydrated substrates, were inoculated in a semi-solid media (C medium). After their incubation at 30 degrees C for 48 hours, the reading device ATB 1520 and the computer of ATB system the reading and automatic interpretation of the results. Using the ATB method, 85 strains were identified (84%) at species level, 9 at genus level and a non-conclusive or unacceptable profile was recorded in 7 strains. From all clinically important yeasts species, a total of 96% were identified by ATB method according to conventional methods. From all non clinically relevant species, ATB 32 C identified correctly 23 strains (78%). ATB 32 C method is a good alternative approach to conventional techniques for identifying yeasts in clinical samples.


[AIDS: descriptive study and survival analysis of the 1st 40 cases]. To analyze the epidemiologic and clinical features, the survival as well as length of stay of the first 40 AIDS cases seen at the internal Medicine Department of the Virgen de la Arrixaca Hospital (Murcia, Spain). Prospective study from September 1988 through July 1991. AIDS criteria used were the CDC (1987) ones. The survival analysis was performed using the Kaplan-Meier method. Thirty-four patients were male (85%) and six female (15%), with a mean age of 39 years. Eighteen (45%) were homo or bisexual, 11 drug addicts (27.5%), 8 (20%) had sexual contacts with prostitutes and/or drug addicts. Two patients were recipients of blood products and in one case no risk could be determined. Opportunistic infections were the first AIDS criteria in 75% of cases. Among the most frequent are P. carinii pneumonia (17.5%), CNS toxoplasmosis (15%) and cryptosporidiosis (12.5%). In 20% of cases a Kaposi's sarcoma developed. The probability of survival after three months was 91% +/- 9.6, at 7 months 79.5% +/- 15.0 and at 15 months 72.5 +/- 18. Overall, 90% of patients had at least one hospital admission, and 27.7% were hospitalized for 50% of more of their survival time. We recorded an increased number of AIDS cases since 1988, being most of them homo-bisexual male patients, with opportunistic infections as first manifestation. We noticed also high proportion of cases of heterosexual transmission.


[Epidemiological factors and vaginal flora changes in vaginal bacteriosis (bacterial vaginosis)]. The aim of this study was to know the modifications of the vaginal bacterial flora that occurs in bacterial vaginosis and to know the involvement of these microorganisms and the influence of several epidemiologic factors in the etiology of this disease. We studied, by using semiquantitative cultures and GLC, vaginal washings from 50 healthy women and 50 women with bacterial vaginosis. The most remarkable results were the high sensitivity of Amsel's criteria and their good correlation with GLC. Women with bacterial vaginosis showed a great increase of CFUs/ml of Gardnerella vaginalis, Mycoplasma hominis, Ureaplasma urealyticum and several species of anaerobic bacteria, and an important decrease of the amounts of aerobic lactobacilli. The main epidemiologic factor among those that were studied was the use of IUDs. The appearance of bacterial vaginosis is associated with the increase of the amounts of G. vaginalis, Bacteroides or related genera (Prevotella, Porphyromonas), and probably M. hominis and U. urealyticum, also being associated to a decrease of the amounts of aerobic lactobacilli. These facts are probably related with alteration in the ecologic relationship lactobacilli/G. vaginalis/anaerobic bacteria.


[Use of PCR in the epidemiological identification of Campylobacter spp]. With arbitrary primer PCR technique it is possible to obtain amplification lane patterns easily and with good reproducibility from the genomic DNA of bacteria studied. There is also no need for prior information regarding the DNA sequence. This method implies two cycles of amplification with low stringency, followed by a PCR of high stringency. Using the above mentioned technique, we were able to show that Campylobacter spp from clinical samples could be separated as well as different strains from the same species. According to our results as well as the ones from different authors applied to other microorganisms, we can assume that the method could be used in any bacterial species for epidemiologic studies purposes.


[Gastroenteritis caused by verotoxin-producing Escherichia coli O157. Presentation of 2 cases]. Verotoxin-producing Escherichia coli strains have been associated with acute hemorrhagic colitis since 1982. We have systematically investigated this pathogen in our laboratory, in all stool samples submitted for culture during a fourteen month period, by using MacConkey sorbitol agar to isolate non sorbitol fermenting Escherichia coli strains. Coated latex particles with an antiserum against Escherichia coli O157 were used to detect O157 serogroup Escherichia coli. A completed serological study and verotoxin assay was performed in all positive strains. We have found two non-related cases of verotoxigenic Escherichia coli infection in two children. In one case, the main clinical picture was an acute hemorrhagic colitis and the other one was a diarrhea without presence of blood, with fever and vomiting. Both cases improved without antimicrobial treatment. No systemic complications appeared in any of the cases during the infection. The infection incidence was 0.07% of all positive stool cultures. The few documented cases of this infection in our country should encourage to investigate this pathogen in order to know its real incidence in our environment.


[Molecular epidemiology of Salmonella enterica poisoning: correlation of the serotype and protein profile and plasmid DNA analysis]. Food-borne diseases nosocomial outbreaks due to Salmonella enterica strains have a high incidence nowadays. We describe here an outbreak that occurred in July 1990 in the Malaga University Hospital, that only involves one shift of health care workers. Salmonella enterica was detected in stool samples from HCW and a first analysis revealed the presence of two different antibiotic susceptibility patterns (resistotypes) among isolated strains. Two months after the outbreak started, the CNVIS (Majadahonda, Spain) confirmed the presence of two different serotypes (bovismorbificans and enteritidis). The delayed availability of the results (due to the lack of specific sera needed in most of the clinical Laboratories) was responsible for finishing only a partial and restricted epidemiologic study in terms of source identification for one particular serotype as well as for the antimicrobial resistance studies. The molecular identification techniques, particularly the electrophoretic protein profile could overcome these problems.


[Resistance of Haemophilus influenzae and Haemophilus parainfluenzae to beta-lactam antibiotics. Characterization of the beta-lactamases]. We have studied the susceptibility to ampicillin and the characteristics of the beta-lactamase activity of the 169 Haemophilus spp. strains (128 H. influenzae, 40 H. parainfluenzae and one H. paraphrophilus) isolated during 12 months, years 1988-1989, in the Hospital del Mar clinical microbiology laboratory. Our objective was to know in the strains of our center the frequency of those resistant or slightly susceptible to ampicillin, the role of beta-lactamases in the loss of susceptibility and the type of enzymes involved. Susceptibility was studied by diffusion for all the antibiotics tested and also confirmed by dilution for ampicillin and other beta-lactam antibiotics. Beta-lactamase production was identified by nitrocefin hydrolysis. The isoelectric point of the beta-lactamase and the identification of their types were determined by analytic isoelectric focusing. The presence of the codifying gene of the TEM-1 enzyme was studied by hybridization with a TEM-1 probe. Of the H. influenzae strains 35 were ampicillin resistant, one moderately susceptible and of the H. parainfluenzae strains six were resistant and two moderately susceptible; all were susceptible to the combination of amoxicillin/clavulanic acid. The ampicillin-resistant strains were beta-lactamase producers. In 40 strains, by isoelectric focusing, the type TEM-1 was identified and the hybridization was positive; in one H. parainfluenzae strain a beta-lactamase of pl 5.8 was observed and the hybridization with TEM-1 probe was negative. The three strains moderately susceptible to ampicillin did not produce beta-lactamase.