Domain closure in mitochondrial aspartate aminotransferase. The subunits of the dimeric enzyme aspartate aminotransferase have two domains: one large and one small. The active site lies in a cavity that is close to both the subunit interface and the interface between the two domains. On binding the substrate the domains close together. This closure completely buries the substrate in the active site and moves two arginine side-chains so they form salt bridges with carboxylate groups of the substrate. The salt bridges hold the substrate close to the pyridoxal 5'-phosphate cofactor and in the right position and orientation for the catalysis of the transamination reaction. We describe here the structural changes that produce the domain movements and the closure of the active site. Structural changes occur at the interface between the domains and within the small domain itself. On closure, the core of the small domain rotates by 13 degrees relative to the large domain. Two other regions of the small domain, which form part of the active site, move somewhat differently. A loop, residues 39 to 49, above the active site moves about 1 A less than the core of the small domain. A helix within the small domain forms the "door" of the active site. It moves with the core of the small domain and, in addition, shifts by 1.2 A, rotates by 10 degrees, and switches its first turn from the alpha to the 3(10) conformation. This results in the helix closing the active site. The domain movements are produced by a co-ordinated series of small changes. Within one subunit the polypeptide chain passes twice between the large and small domains. One link involves a peptide in an extended conformation. The second link is in the middle of a long helix that spans both domains. At the interface this helix is kinked and, on closure, the angle of the kink changes to accommodate the movement of the small domain. The interface between the domains is formed by 15 residues in the large domain packing against 12 residues in the small domain and the manner in which these residues pack is essentially the same in the open and closed structures. Domain movements involve changes in the main-chain and side-chain torsion angles in the residues on both sides of the interface. Most of these changes are small; only a few side-chains switch to new conformations.(ABSTRACT TRUNCATED AT 400 WORDS)


Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution. The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.


Topology fingerprint approach to the inverse protein folding problem. We describe the most general solution to date of the problem of matching globular protein sequences to the appropriate three-dimensional structures. The screening template, against which sequences are tested, is provided by a protein "structural fingerprint" library based on the contact map and the buried/exposed pattern of residues. Then, a lattice Monte Carlo algorithm validates or dismisses the stability of the proposed fold. Examples of known structural similarities between proteins having weakly or unrelated sequences such as the globins and phycocyanins, the eight-member alpha/beta fold of triose phosphate isomerase and even a close structural equivalence between azurin and immunoglobulins are found.


Structure of scorpion toxin variant-3 at 1.2 A resolution. The crystal structure of the variant-3 protein neurotoxin from the scorpion Centruroides sculpturatus Ewing has been refined at 1.2 A resolution using restrained least-squares. The final model includes 492 non-hydrogen protein atoms, 453 protein hydrogen atoms, eight 2-methyl-2,4-pentanediol (MPD) solvent atoms, and 125 water oxygen atoms. The variant-3 protein model geometry deviates from ideal bond lengths by 0.024 A and from ideal angles by 3.6 degrees. The crystallographic R-factor for structure factors calculated from the final model is 0.192 for 17,706 unique reflections between 10.0 to 1.2 A. A comparison between the models of the initial 1.8 A and the 1.2 A refinement shows a new arrangement of the previously poorly defined residues 31 to 34. Multiple conformations are observed for four cysteine residues and an MPD oxygen atom. The electron density indicates that disulfide bonds between Cys12 and Cys65 and between Cys29 and Cys48 have two distinct side-chain conformations. A molecule of MPD bridges neighboring protein molecules in the crystal lattice, and both MPD enantiomers are present in the crystal. A total of 125 water molecules per molecule of protein are included in the final model with B-values ranging from 11 to 52 A2 and occupancies from unity down to 0.4. Comparisons between the 1.2 A and 1.8 A models, including the bound water structure and crystal packing contacts, are emphasized.


Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells. The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.


Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme. The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.


Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha. The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.


Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution. A target enzyme for chemotherapy. The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution. The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map. The enzyme forms a dimer in the crystal structure. Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet. The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand. A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue. The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme. GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis. Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs.


The Escherichia coli rpoB60 mutation blocks antitermination by coliphage HK022 Q-function. The lambdoid bacteriophage regulate gene expression by suppressing transcription terminators. Although similar in sequence to lambda, HK022 lacks an analogue to the lambda N antitermination gene and a distinct nutR sequence. To define the HK022 antitermination system, we plated the phage on Escherichia coli nus mutants that inhibit lambda N function. Only rpoB60 (also called nusC60) blocked HK022 lytic growth. Analyses of HK022-lambda hybrid phage suggested that a HK022 function analogous to lambda Q was inhibited by rpoB60. This result was confirmed with pR'-tR'-galK fusions. HK022 Q-protein suppressed tR' in wild-type but not in rpoB60 mutants. The lambda Q-protein, although inhibited by rpoB60, was more active than the HK022 analogue. A single amino acid difference between the two Q-proteins accounts for the phenotype. Changing the penultimate residue of HK022 Q from alanine to the lambda threonine generated a phage that could propagate on rpoB60 hosts. Host and phage mutations that permitted HK022 growth in rpoB60 strains were characterized. The bacterial suppressors were located in the Escherichia coli nusB gene. The phage suppressors represented recessive mutations in a HK022 b-region sequence encoding an open reading frame of 73 codons.


Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates. The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.


Site-directed mutagenesis of the GTP-binding domain of beta-tubulin. Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.


Active site of trypanothione reductase. A target for rational drug design. The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.


Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible. We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.


Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli. Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.


Crystallization of a type I 3-dehydroquinase from Salmonella typhi. Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.


Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila. The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.


Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein. Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.


Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli. Circular dimer plasmids linearized with a restriction endonuclease undergo intramolecular recombination to yield recombinant circular monomers at high efficiency by a recA-independent mechanism in Escherichia coli recB recC sbcA mutants. The rate of this reaction is at least 1000-fold higher than the recombination rate observed for circular plasmid recombination substrates in the same mutants. Three potential models have been previously proposed to explain the recombination events observed. The validity of these models was tested in recA recB recC sbcA mutants using additional recombination substrates. These substrates, when linearized by incubation with an appropriate restriction enzyme, contain non-homologous adenovirus 2 DNA on one or both ends. The data indicate that terminal non-homology does not significantly affect the efficiency of recovering recombinants. In contrast to many recombination models proposed that involve the invasion of homologous duplex DNA by single-stranded DNA ends, the intramolecular recombination reaction studied here does not appear to involve direct pairing from the end(s) of the substrate DNA. Furthermore, the results are consistent with a model proposing that pairing and strand exchange occur between two homologous duplex regions within the linear dimer molecule.


Alpha-tubulin gene family of maize (Zea mays L.). Evidence for two ancient alpha-tubulin genes in plants. Among 81 alpha-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different alpha-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different alpha-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93%. The results of hybridizations of genomic DNAs to alpha-tubulin coding region probes and to 3' non-coding region probes constructed from six different alpha-tubulin cDNA clones indicated that the maize alpha-tubulin gene family contains at least eight members. Comparison of deduced alpha-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that alpha-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient alpha-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily II genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.


1H nuclear magnetic resonance studies of the interaction of urea with hen lysozyme. Origins of the conformational change induced in hen lysozyme by N-acetylglucosamine oligosaccharides. The interaction between hen lysozyme and urea has been investigated using 1H nuclear magnetic resonance spectroscopy. Chemical shift changes for resonances of a number of residues in the vicinity of the active site of the protein have been observed in the presence of urea prior to denaturation. These shifts are similar to those induced in the hen lysozyme spectrum by the specific binding of N-acetylglucosamine (GlcNAc) in site C of the active site cleft, indicating that urea and GlcNAc induce a similar conformational change in the enzyme. This implies that the conformational changes experienced by the enzyme on the binding of GlcNAc oligosaccharides are the consequence of interactions, possibly hydrogen bonding, involving the N-acetyl group of the sugar residue bound in site C, rather than the result of contacts between the protein and the pyranose rings of the oligosaccharides. This suggests that hen lysozyme employs an induced fit type mechanism to discriminate for N-acetylated saccharides as substrates.


Tubulin gene expression in maize (Zea mays L.). Change in isotype expression along the developmental axis of seedling root. Two-dimensional gel/western blot analysis was used to characterize alpha- and beta-tubulin isotype expression along the developmental axis of the maize (Zea mays) seedling primary root. We identified four distinct alpha-tubulin isotypes and a minimum of six beta-tubulin isotypes. This analysis showed differences between the alpha- and beta-tubulin isotypes expressed in rapidly dividing tissue at the root tip and differentiated root tissues proximal to the tip. The alpha 1 and alpha 4 isotypes predominated in samples from immature rapidly dividing tissues such as root tips, whereas in mature tissues such as differentiated root and pollen, alpha 2, alpha 3 and alpha 4 isotypes predominated. The beta 1 and beta 2 isotypes were more abundant in protein samples from root cortex than in samples from the root tip or vascular cylinder. In contrast, the beta 4 and beta 5 isotypes appeared to be more abundant in root tip and vascular cylinder samples than in root cortex samples. Hybridization probes from the 3' non-coding region of six alpha-tubulin cDNA clones were used to quantify the levels of corresponding tubulin transcripts in selected tissues, from embryonic to mature and from largely undifferentiated to highly differentiated. The results from these hybridization experiments showed that all of the alpha-tubulin genes were expressed in all tissues examined, although each gene showed a unique pattern of differential transcript accumulation. A transcript produced from cDNA clone representing the tua5 alpha-tubulin gene was translated in vitro and produced an alpha-tubulin that comigrated with the alpha 2 isotype.


Inhibition of benzo[a]pyrene-induced bone marrow micronuclei formation by diallyl thioethers in mice. Diallyl thioethers (DATEs), naturally occurring compounds present in garlic, were investigated for their putative ability to inhibit benzo[a]pyrene-induced genotoxicity in ICR and C3H strains of mice. The mouse bone marrow micronucleus assay was used as an indicator of in vivo genotoxicity. A dose of 0.67 mmol total DATEs/kg body weight inhibited formation of micronucleated polychromatic erythrocytes (MPCEs) by 24%, and 0.33 mmol DATEs inhibited formation of MPCEs by 45%. Possibly the toxicity of DATEs accounted for less inhibition with the higher dose. Formation of MPCEs were inhibited only slightly by DATEs in C3H mice. These results indicate that the mouse bone marrow micronucleus assay can be used to identify organosulfur components of garlic that inhibit genotoxicity.


Dichloroacetic acid and trichloroacetic acid increase chloroform toxicity. Dichloro- and trichloroacetic acids (DCA and TCA) and chloroform are formed during chlorination disinfection of drinking water. The effects of DCA and TCA treatment on CHCl3 toxicity were assessed in these studies. Male and female rats were gavaged with DCA or TCA (0.92 and 2.45 mmol/kg administered 3 times over 24 h). Three hours after the last dose CHCl3 was injected ip (0.75 mg/kg). Male rats experienced some weight loss (15%) and slight increases of ALT and BUN, but there were no effects of either DCA or TCA on any of these responses. In females, CHCl3 increased plasma ALT and this response was greater (up to threefold) in the DCA group, compared to saline controls. Similarly, BUN was increased by CHCl3 and this was more severe (up to threefold) in both the DCA and TCA pretreated groups. These results show that CHCl3 toxicity is increased by DCA and TCA, and this effect is gender-specific, occurring only in females. DCA increases both liver and kidney toxicity, whereas TCA affects only kidney toxicity.


Toxicity assessment of atrazine, alachlor, and carbofuran and their respective environmental metabolites using Microtox. Using the Microtox method of toxicity assessment designed by Microbics Corporation, the relative toxicities of alachlor, atrazine, and carbofuran, three pesticides commonly used in agricultural production, were determined. Generally, carbofuran was found to be most acutely toxic, followed closely by atrazine. Alachlor was least toxic of the three pesticides tested. Selected environmental metabolites of these three agri-chemicals were also tested using the same method. Hydroxyalachlor, deethylatrazine, deisopropylatrazine, 3-hydroxycarbofuran, and 3-ketocarbofuran were selected for analysis because previous studies determined their presence in surface and ground-water supplies along with their parents. Results showed that often the metabolites were at least as acutely toxic as their parents, particularly in the case of 3-ketocarbofuran and hydroxyalachlor, which demonstrated toxicities higher or not significantly different than their parents. Hydroxycarbofuran was assessed as the least toxic of all substances tested. The atrazine environmental metabolites were less toxic than their parent.


Hepatotoxic interactions of ethanol with allyl alcohol or carbon tetrachloride in rats. To assess whether potential toxic interactions occur between ethanol and allyl alcohol or carbon tetrachloride following subacute, concurrent chemical exposure, male Fischer 344 rats, approximately 70 d of age, were given ethanol at 0, 0.05, 0.1, 0.2, or 0.5 ml/kg in corn oil daily by gavage for 14 d (ETOH group), or the same levels of ethanol with 21 mg allyl alcohol/kg (ALAC group), or the same levels of ethanol with 20 mg carbon tetrachloride/kg (CCL4 group). Hepatic response was assessed 24 h after the last dose. Interactions were evaluated by comparing the ETOH group with either the ALAC group or the CCL4 group using multivariate analysis of variance procedures. No statistically significant interaction was seen between the ETOH group and the ALAC group at the dosages used. Although an interaction between ethanol and carbon tetrachloride given simultaneously was not statistically significant, a small interactive effect on weight gain from d 0 to termination was apparent (p = .057). Exposure to ethanol alone resulted in a concentration-dependent decrease in absolute and relative liver weight, with a threshold between 0.05 and 0.1 ml/kg. There was no histopathological evidence of hepatic damage with ethanol alone, and no effect on hepatic cytochrome P-450 and glutathione levels or on serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALK). Exposure to allyl alcohol alone resulted in significant increases in absolute and relative liver weights, liver glutathione, and periportal hepatocellular vacuolar degeneration. Exposure to carbon tetrachloride alone resulted in significant increases in absolute and relative liver weight, serum levels of ALT, AST, and ALK, and centrilobular hepatocellular vacuolar degeneration and necrosis. These observations indicate that subacute, concurrent exposure of ethanol with carbon tetrachloride or allyl alcohol at ethanol levels comparable to those reported in gavage vehicles did not result in interactive toxicity.


Review of the allergic contact dermatitis hazard posed by chromium-contaminated soil: identifying a "safe" concentration. At least 200 sites in the United States contain soil with elevated levels of trivalent and hexavalent chromium [Cr(III) and Cr(VI)]. Although the potential cancer hazard posed by airborne Cr(VI) has been the primary concern for these sites, a soil cleanup standard based on the potential elicitation of allergic contact dermatitis has been proposed for sites in Hudson County, N.J. This paper describes the rationale for identifying a soil concentration of Cr(VI) that should not pose an allergic contact dermatitis hazard-even in sensitized persons. A literature review of eight published patch test studies that evaluated the allergic response to potassium dichromate was conducted. These studies were evaluated for clinical and statistical relevance in establishing a threshold dose of Cr(VI) to which no more than 10% of the subpopulation sensitized to chromium would respond, and that would protect at least 99.84% of the general population. Although each of the studies had certain methodological limitations when evaluated against current test methods, the data set proved useful for deriving an estimated threshold. Using computer data-fitting techniques based on truncated lognormal distributions, a weighted mean 10% threshold of approximately 150 ppm potassium dichromate or 54 ppm Cr(VI) was identified for the eight studies. Due to the types of limitations noted for these studies, this threshold is likely to be somewhat conservative. Test results have shown that between 5 and 10% of the Cr(VI) at concentrations less than about 500 ppm are released from a soil matrix into an isotonic saline solution simulating sweat. Using human sweat as the extractant, it has been shown that only 0.1% of the CR(VI) at concentrations of approximately 1,000 ppm are released from a soil matrix into sweat. Based on 10% solubilization of soil-bound Cr(VI) and the results of our statistical analysis of previous threshold studies, a concentration of approximately 350 to 500 ppm Cr(VI) in soil should be sufficiently low to protect virtually all exposed people, including children, from chromium-induced allergic contact dermatitis.


Role of oxidative stress in single-dose, cadmium-induced testicular cancer. Treatment of rats with a single carcinogenic dose of CdCl2 (i.e., 30 mumol/kg) caused severe hemorrhagic damage in the testis within the first 12 h after the metal. Subsequently, atrophy with calcification developed in the next 2-3 mo. Atrophied tissues regenerated during the 1 yr after exposure. Twelve hours after exposure to the Cd treatment, lipid peroxidation levels, Fe content, and cellular production of H2O2 were remarkably elevated in testicular Leydig cells, the target cell population for Cd carcinogenesis. At the same time, glutathione peroxidase activity rose, glutathione reductase and catalase activities were reduced, and superoxide dismutase activity was unchanged. Xanthine oxidase activity in Leydig cells was also elevated at 6 and 9 h after the Cd treatment. Reduced glutathione in testes was decreased and oxidized glutathione was increased 12 h after exposure to the metal. These facts suggest that the carcinogenic doses of Cd induced oxidative stress while compromising cellular defense mechanisms against such stress. Therefore, active oxygen species such as H2O2 may have an important role in the initiation of carcinogenesis within the target cell population.


Urethanase in rat tissues.

Urethanase in rat tissues. Urethane-hydrolyzing activity (urethanase) was found in the homogenates of liver, kidney, and lung of rats. The activity was significantly higher at acidic condition (pH 5.0) than that at neutral condition (pH 7.0) in every case. The highest activity among them was observed in a kidney homogenate preparation. The activities increased slightly with treatment by Triton X-100. Intracellular distribution of urethanase in rat kidney was examined and found that the enzyme activity located mainly in the lysosomal fraction. The optimal pH was found to be 5.0. The enzyme activity was strongly inhibited by EDTA, whose 50% inhibitory concentration was 7.4 x 10(-7) M. Complete loss of the enzyme activity by the addition of EDTA was fully recovered by the addition of Zn2+, which suggested that urethanase belongs to the category of Zn enzyme.


Embryotoxic and teratogenic effects of intraperitoneally administered metavanadate in mice. Metavanadate was evaluated for developmental toxicity in pregnant Swiss mice. Sodium metavanadate (NaVO3) was administered intraperitoneally on d 6-15 of gestation at doses of 0, 2, 4, or 8 mg/kg/d. On gestation d 18, all live fetuses were examined for external, visceral, and skeletal malformations and variations. Maternal toxicity was observed at 2, 4, and 8 mg/kg/d as evidenced by decreased weight gain during treatment. Increased resorptions and dead fetuses, increased percentage postimplantation loss, and reduced fetal body weight per litter were observed at 4 and 8 mg/kg/d. There were no significant increases in the type or incidence of external and skeletal anomalies, but a significant increase in the incidence of cleft palate was detected at 8 mg/kg/d. The lowest-observed-adverse-effect level (LOAEL) for maternal toxicity was 2 mg NaVO3/kg/d, while 2 mg/kg/d was also the no-observed-adverse-effect level (NOAEL) for significant developmental toxicity.


Dermal absorption of chemicals: effect of application of chemicals as a solid, aqueous paste, suspension, or in volatile vehicle. The purpose of this study was to investigate the dermal absorption of chemicals in different physical forms when applied to female F344 rats. Chemicals were applied either as a solid, aqueous paste, suspension, or dissolved in the volatile vehicle ethanol. The chemicals investigated were [14C]-2-sec-butyl-4,6-dinitrophenol (DNBP, 4.2 mumol), 2,4,5,2',4',5'-[14C]-hexachlorobiphenyl (HCB, 2.3 mumol), and 3,4,3',4'-[14C]-tetrachlorobiphenyl (TCB, 0.5 mumol). The chemicals were applied on the clipped mid-dorsal region of the rat over a 2.54-cm2 treatment area, which was then occluded. Urine and feces were collected and assayed for radioactivity. Twenty-four hours post-application, the treated skin was washed with a mixture (1:1) of soap and water, dried, and reoccluded. The animals were sacrificed at 120 h by exsanguination under ether anesthesia. Radioactivity in the blood, skin (treated and untreated), and carcass was assayed. Dermal absorption of DNBP-derived radioactivity was approximately 50% of the recovered dose after application in the four physical forms, and the major route of excretion was via the urine. Twelve percent of the absorbed dose of DNBP was retained in the body. Dermal penetration of HCB-derived radioactivity was 5-8% of the recovered dose after application in the four forms, and the major route of excretion was via the feces. Greater than 90% of the absorbed dose of HCB-derived radioactivity was retained in the body. Dermal penetration of TCB-derived radioactivity was 6-8% of the recovered dose in the four forms, and the major route of excretion was via the feces. Approximately 21% of the absorbed dose was retained in the body at 120 h. Absorption of each chemical applied either as solid, aqueous paste, or suspension was compared to the absorption of the same chemical in ethanol. Absorption of HCB applied as a solid was significantly higher (p less than or equal to .05) as compared to HCB applied in ethanol. There were no other significantly differences in the comparisons of absorption. The data indicate that the chemicals examined in this study can penetrate the skin as readily when applied either as a solid, aqueous paste, or suspension, as when applied in the volatile vehicle ethanol.


PCDD and PCDF in human milk from Scandinavia, with special emphasis on Norway. Levels of polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were measured in human milk in a collaborative Scandinavian study with 10 samples from each of 7 locations in Norway and Sweden (total 70 samples) and 10 samples from Denmark. Four locations represented areas with different PCDD and PCDF sources and three background areas. Norwegian results are presented and related to the Danish and Swedish values. No geographical differences were found in total dioxins expressed as Nordic TCDD equivalents. Mean equivalent values (pg/g fat basis) were 15-18 in Norway and Denmark and 20-24 in Sweden, lower than those reported from parts of Western Europe but higher than in the undeveloped countries. When looking at the individual compounds, the highest values of PCDD congeners were observed in industrialized areas of Sweden, while the highest PCDF values were found in a Norwegian area known for dioxin contamination from a magnesium-producing factory.


Toxicokinetics of mercuric chloride and methylmercuric chloride in mice. Future human exposure to inorganic mercury will probably lead to a few individuals occupationally exposed to high levels and much larger populations exposed to low or very low levels from dental fillings or from food items containing inorganic mercury; human exposure to methylmercury will be relatively low and depending on intake of marine food. Ideally, risk assessment is based on detailed knowledge of relations between external and internal dose, organ levels, and their relation to toxic symptoms. However, human data on these toxicokinetic parameters originate mainly from individuals or smaller populations accidentally exposed for shorter periods to relatively high mercury levels, but with unknown total body burden. Thus, assessment of risk associated with exposure to low levels of mercury will largely depend on data from animal experiments. Previous investigations of the toxicokinetics of mercuric compounds almost exclusively employed parenteral administration of relatively high doses of soluble mercuric salts. However, human exposure is primarily pulmonary or oral and at low doses. The present study validates an experimental model for investigating the toxicokinetics of orally administered mercuric chloride and methylmercuric chloride in mice. Major findings using this model are discussed in relation to previous knowledge. The toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex, whereas the mouse strain used is less important. The "true absorption" of a single oral dose of HgCl2 was calculated to be about 20% at two different dose levels. Earlier studies that did not take into account the possible excretion of absorbed mercury and intestinal reabsorption during the experimental period report 7-10% intestinal uptake. The higher excretion rates observed after oral than after intraperitoneal administration of HgCl2 are most likely due to differences in disposition of systemically delivered and retained mercury. After methylmercury administration, mercury excretion followed first-order kinetics for 2 wk, independently of administration route, strain, or sex. However, during longer experimental periods, the increasing relative carcass retention (slower rate of excretion) caused the elimination to deviate from first-order kinetics. Extensive differences in the toxicokinetics of methylmercury with respect to excretion rates, organ deposition, and blood levels were observed between males and females.(ABSTRACT TRUNCATED AT 400 WORDS)


Assessment of the empty follicle syndrome by transvaginal sonography. The empty follicle syndrome was assessed using transvaginal ultrasonography in a group of 152 consecutive women with unmedicated menstrual cycles being studied because of primary or secondary infertility or repetitive miscarriage. The overall frequency of the empty follicle syndrome was found to be 43.4%. The frequency increased with age but was independent of gravidity. The empty follicle syndrome may be a significant etiologic factor in infertility or other reproductive abnormalities, and transvaginal ultrasound represents a good, non-invasive means of evaluating it.


The effect of altitude on the umbilical artery Doppler resistance. This study examined whether there were differences between the umbilical artery systolic-diastolic (S/D) ratio and resistance index (RI) in patients with normal fetuses living at sea level (group I) and those living between 4,200 and 4,500 feet above sea level (group II). Linear regression demonstrated a significant association (P less than 0.001) between gestational age and the S/D ratio and RI for groups I (n = 100) and II (n = 128). Least-squares regression analysis demonstrated no significant differences in slopes or intercepts between groups I and II. The results would suggest that altitude, up to 4,500 feet above sea level, does not affect umbilical artery Doppler resistance in normal fetuses.


First trimester findings in pregnancies after in vitro fertilization. We assessed the frequency of abnormal sonographic findings and their significance with respect to outcome in pregnancies resulting from in vitro fertilization (IVF). We retrospectively reviewed first trimester sonograms of 53 consecutive IVF patients who had a positive pregnancy test and first trimester ultrasonography at least 4 weeks after embryo transfer, and we correlated the sonographic findings with pregnancy outcome. For controls, we compared the frequency of sonographic abnormalities in these study patients to that in a group of patients who became pregnant after ovulation induction only. In the 53 IVF patients, the numbers of gestational sacs identified on the initial sonogram were as follows: 34 singletons, 11 twins, two triplets, one quadruplets and one quintuplets; no sac was seen in four patients. In 32 patients, the first sonogram was normal, with a visualized yolk sac, or heartbeat, or both. In 10 patients the gestational sac appeared abnormal but sac contents were normal. In seven patients an abnormality of sac contents was identified, including four with an embryo but no heartbeat and three anembryonic sacs. Overall, 40% of IVF patients had sonographic abnormalities, in comparison to 7% in the control group of patients (P less than 0.05, Fisher's exact test). Of the 32 patients with normal sonograms, 26 (81%) delivered at least one live infant. Of the 10 patients whose gestation sacs appeared abnormal, nine (90%) gave birth to live infants. Of the seven patients with abnormal sac contents, two (29%) delivered at least one live infant. We conclude that abnormal findings are frequently present on the initial sonogram of pregnant patients after IVF.(ABSTRACT TRUNCATED AT 250 WORDS)


A comparison of duplex Doppler sonography of the ligamentum teres and portal vein with endoscopic demonstration of gastroesophageal varices in patients with chronic liver disease or portal hypertension, or both. The ability of duplex Doppler sonography of the ligamentum teres and portal vein to detect specific signs of portal hypertension was compared with the ability of endoscopy to demonstrate gastroesophageal varices in consecutive patients. Among 90 patients with parenchymal liver disease and a high probability of portal hypertension, 70 had varices, 72 had specific sonographic signs, and four had neither. Ultrasonography was comparable to endoscopy irrespective of the clinical severity of the underlying liver disease. Eleven patients had vascular occlusive diseases; nine had varices; and all had at least one sonographic sign. Duplex Doppler ultrasonography may have a clinical role in noninvasive detection of portal hypertension. Further studies correlating the findings with those of portal pressure are needed to define the place of duplex Doppler ultrasonography as a predictor of the presence of portal hypertension.


Placenta accreta: prospective sonographic diagnosis in patients with placenta previa and prior cesarean section. A prospective evaluation for possible placenta accreta was performed in 34 patients with placenta previa and a history of one or more cesarean sections. Sonographic criteria used included (1) loss of the normal hypoechoic retroplacental myometrial zone, (2) thinning or disruption of the hyperechoic uterine serosa-bladder interface, and (3) presence of focal exophytic masses. Of 18 patients with positive sonographic results, 14 had proof of placenta accreta and 16 of the patients underwent hysterectomy. Of 16 patients with negative sonographic results, only one had placenta accreta, and two patients required hysterectomy. Presence of numerous intraplacental vascular lacunae appears to be an additional risk criterion for placenta accreta, separate from the other criteria listed above.


The aortic and pulmonary outflow tract screening examination in the human fetus. Imaging of the aortic and pulmonic outflow tracts in the fetus may be difficult owing to fetal position. This study describes two screening methods for imaging the outflow tracts, depending on whether the interventricular septum was tangential (group I) or perpendicular (group II) to the ultrasonic beam. We performed 170 examinations during the second and third trimesters of pregnancy. The outflow tracts were imaged in 166 (97.6%). Fetuses in group I were identified much less frequently (13.2%) than fetuses in group II (86.8%). This study would suggest that for the outflow tracts to be imaged during a screening examination, the sonographer should become familiar with the sonographic anatomy of the outflow tracts when the interventricular septum is perpendicular to the ultrasound beam.


Tubular ectasia within the mediastinum testis. Eleven scrotal sonographic examinations showing a spectrum of findings within the mediastinum testis were collected over a 2 year period. Each case showed numerous small tubular or rounded anechoic structures within the mediastinum testis; often, the findings mimicked a hypoechoic mass. Findings were bilateral in eight of ten patients; one additional patient had only one testis because of orchiectomy. All patients had an associated extratesticular finding, in most cases a spermatocele. Tubular ectasia shares several features with testicular cysts and mechanisms of formation are postulated to be similar to those previously proposed for testicular cysts. Recognizing tubular ectasia is important to avoid unnecessary concern and potential surgery.


Contemporary surgical management of renovascular disease. To examine the treatment methods and early results of renovascular repair in our contemporary patient population, we reviewed our surgical experience during a recent 54-month period. From January 1987 to July 1991, 200 patients ranging in age from 5 to 80 years (mean, 56 years) were operated on for correction of nonatherosclerotic (43 patients) and atherosclerotic (157 patients) renovascular disease. The group included 92 men and 108 women, with blood pressures ranging from 300/198 mm Hg to 120/70 mm Hg (mean, 205/113 mm Hg). Defined by preoperative serum creatinine, 129 patients (65%) had evidence of renal insufficiency (Cr greater than or equal to 1.3 mg/dl), whereas 71 patients (36%) had severe renal insufficiency (Cr greater than 2.0 mg/dl; 11 patients were dependent on dialysis). One hundred forty-seven patients with atherosclerotic renovascular disease (94%) demonstrated organ-specific atherosclerotic damage. Operative management of 291 kidneys included unilateral renal artery repair in 117 patients (58%), bilateral repair in 78 patients (39%), and primary nephrectomy in five patients (2.5%). Simultaneous aortic reconstruction was required in 64 patients (32%). There were five operative deaths (2.5% mortality rate) and four occluded renovascular repairs (1.4% primary failure) within 30 days of surgery. Hypertension was considered cured in 21% and improved in 70% of 195 operative survivors. In 70 patients with severe renal insufficiency before operation, estimated glomerular filtration rate was improved in 49% (8 of 11 patients removed from dialysis), unchanged in 36%, and worsened in 15%. Renal function response was significantly influenced by the site of disease and the operation. Twenty-six additional postoperative deaths occurred during follow-up (range, 6 to 58 months; mean, 24.4 months). Extreme atherosclerotic-renovascular disease, preoperative renal insufficiency, failure to improve renal function, and progression to dependence on dialysis after operation were associated with follow-up deaths. Although most patients had a beneficial outcome, failure to improve extreme renal insufficiency was associated with a rapid rate of death during a relatively short follow-up period.


Penetrating abdominal aortic trauma: a report of 129 cases. The clinical presentation, resuscitation, and operative management of 129 patients with penetrating injuries to the abdominal aorta treated between 1960 and 1989 were reviewed. This is the largest reported civilian or military experience with this specific injury; our review of the literature was limited to reports focusing on the detailed analysis of the management and outcome of aortic trauma. Eighty-two percent of patients were in shock; 18 patients (14%) underwent emergency room thoracotomies, with no survivors. Of 46 patients requiring operating room thoracotomies, only 20% survived. All patients underwent exploration. Thirty percent had three or more visceral injuries; only 2% had no concurrent visceral injury. There were associated vascular injuries in 58% of patients, the inferior vena cava being the most frequent (37%). The most significant predictor of death was continued bleeding at operation. Ninety percent of patients with free intraperitoneal bleeding died, in contrast to 35% with a contained retroperitoneal hematoma (p less than 0.001). Aortic injuries were supraceliac in 25% of patients, between the celiac and renal arteries in 25%, and infrarenal in 50%. The respective mortality rates were 70%, 80%, and 47% (p less than 0.05). Repair of the aortic defect was possible in 103 patients (80%). The most common repair was lateral arteriorrhaphy in 53% of patients, followed by end-to-end anastomosis in 15% and prosthetic repairs in 8%. The overall mortality rate was 62%, which is similar to that of previous reports. Despite advances in trauma care, a functioning paramedic system, and the use of aggressive means to obtain aortic control, penetrating aortic trauma remains highly lethal.


Are gram-negative bacteria a contraindication to selective preservation of infected prosthetic arterial grafts? Management of infected prosthetic arterial grafts has traditionally included total graft excision especially when gram-negative bacteria were cultured. Between 1973 and 1991 we treated 42 patients with infected prosthetic grafts (33 polytetrafluoroethylene (PTFE), 9 Dacron) by complete graft preservation when the graft was patent, the anastomoses were intact, and the patient did not have sepsis. The infection involved the anastomosis (36 cases) or the body (6 cases) of 33 peripheral grafts and the distal segment of five aortofemoral and four iliac-distal grafts. Cultures of the 42 infected grafts grew gram-positive bacteria in 33 cases and gram-negative bacteria in 22 cases. Treatment adjuncts included repeated, radical operative wound debridement and rarely (7 of 42) rotational muscle flaps. This management resulted in a 10% (4 of 42) hospital mortality rate and an amputation rate in survivors of 3% (1 of 38 threatened limbs). All four deaths were due to sepsis: gram-positive bacteria were cultured in all cases and gram-negative bacteria in two cases. Of the 38 survivors, 29 (76%) wounds healed and remained healed after average follow-up of 3 years (range, 1 to 18 years). Nine other patients required total graft excision for nonhealing wounds (7 cases) or delayed anastomotic hemorrhage (2 cases). Gram-negative bacteria were cultured in four, and gram-positive bacteria were cultured in six of these nine wounds. Four of nine (44%) graft infections that cultured Pseudomonas organisms healed without complications versus 23 of 33 (70%) wounds that cultured gram-positive bacteria, and 12 of 13 (92%) wounds that cultured gram-negative bacteria other than Pseudomonas organisms.(ABSTRACT TRUNCATED AT 250 WORDS)


Does contralateral internal carotid artery occlusion increase the risk of carotid endarterectomy? The results of every carotid endarterectomy performed contralateral to an internal carotid artery occlusion (n = 36) (group I) were compared with those performed contralateral to a patent internal carotid artery (n = 169) (group II) over the last 10 years. The patients in each group were evenly matched with respect to male gender (66% vs 69%); mean age (66.7 vs 65.9 years); and incidence of hypertension (55.6% vs 53.2%), diabetes (16.7% vs 20.1%), and hyperlipidemia (8.3% vs 11.8%). Patients in group I had a higher incidence of previous myocardial infarction (25% vs 11.8%, p less than 0.05) and exertional angina (55.6% vs 29.6%, p less than 0.01). Indications for carotid endarterectomy were equivalent, including stroke (19.4% vs 21.9%), transient ischemic attacks (36.1% vs 35.5%), amaurosis fugax (16.7% vs 11.8%), nonhemispheric symptoms (5.6% vs 8.3%), and asymptomatic stenoses (22.2% vs 22.5%), respectively. Perioperative strokes occurred in one (2.8%) patient in group I and seven (4.1%) patients in group II (NS). Among the patients in group II the incidence of perioperative stroke did not correlate directly with the degree of contralateral ICA stenosis: greater than 90% (4%); 70% to 90% (6.7%); 50% to 70% (8.7%); and less than 50% (2.8%). The operative mortality rate was 0% among patients in group I and 1.2% among patients in group II (NS). Cardiac complications occurred in two (5.6%) patients in group I and nine (5.3%) patients in group II (NS).(ABSTRACT TRUNCATED AT 250 WORDS)


The evolving surgical management of recurrent carotid stenosis. The traditional approach to recurrent carotid stenosis has been repeat endarterectomy or patch angioplasty. Concern with the durability of repeat carotid endarterectomy has resulted in our use of carotid resection with autogenous graft interposition. This study was designed to determine the outcome and efficacy of carotid resection compared with repeat carotid endarterectomy in the management of recurrent carotid stenosis. From 1974 to 1991, 162 operations (repeat carotid endarterectomy 105, carotid resection 57) were performed for recurrent carotid stenosis. Indication for operation was hemispheric symptoms in 63% of patients, nonlateralizing symptoms in 25%, asymptomatic stenosis in 7%, and previous stroke in 5%. Ninety-one percent of patients had stenosis greater than 90% on arteriography. The perioperative stroke rate for carotid resection was 3.5%, with a subsequent rate of 0.0064 strokes per year. For repeat carotid endarterectomy, the perioperative stroke rate was 1.9% with a subsequent rate of 0.011 strokes per year. Graft patency after carotid resection was 93% (mean follow-up, 35 months). Four patients treated with carotid resection had graft thrombosis, and two of the four remained asymptomatic. After repeat carotid endarterectomy, one patient had carotid thrombosis, and recurrent stenosis greater than 50% developed in 23 patients (mean follow-up, 64 months). Twenty patients treated with repeat carotid endarterectomy underwent an additional operation for further symptomatic recurrent carotid stenosis. We conclude carotid resection is a safe and effective alternative to repeat carotid endarterectomy for patients undergoing operation for recurrent carotid stenosis.


Maffucci's syndrome (hemangiomatosis osteolytica): a report of four cases. Maffucci's syndrome is a congenital nonfamilial syndrome combining dyschondroplasia, (enchondromatosis) and hemangiomatosis. It is a rare disease; only 200 cases have been reported throughout the world in the past 140 years. Over the past 20 years, four patients have been admitted with signs and symptoms consistent with Maffucci's syndrome. Three were children ages 3, 7, and 9 years. The fourth was 23 years old. Two were male and two female. All had hemangiomas at birth, and all had skeletal deformities and enchondromas. All complained of pain and heaviness of the involved extremity. Three patients had the arterial inflow evaluated with arteriograms, and one had magnetic resonance imaging. Two also had venograms. Two patients had excision of their hemangiomas, and one had sclerotherapy and compression therapy. All had bone biopsies performed. None of the enchondromas or the soft tissue lesions had undergone sarcomatous transformation.


Superiority of end-diastolic volume and ejection fraction measurements over wedge pressures in evaluating cardiac function during aortic reconstruction. Marked changes occur in cardiac function during aortic reconstruction. Numerous factors (primarily compliance changes) limit the currently used pressure-based pulmonary artery catheters' ability to reflect these changes accurately. A new pulmonary artery catheter was used in 26 patients undergoing aortic reconstruction (7 patients with abdominal aortic aneurysms and 19 with occlusive disease) that directly measures right ventricular (RV) volumes and ejection fractions. The extent of coronary artery disease (CAD) was evaluated before surgery, and the patients were divided into three classes. The greatest changes in cardiac function were noted immediately after aortic cross-clamping. Cardiac output significantly decreased in patients with mild or moderate CAD (p less than 0.01 and p less than 0.001) but not in patients with no CAD (p less than 0.5). This correlated well with RV end-diastolic volume (RV-EDV) and stroke volume (RV-SV) measurements in the groups with mild (p less than 0.05) and moderate CAD (p less than 0.01) but not in the group with no CAD (p greater than 0.5). Wedge pressure showed poor correlation, with no significant change noted in any group (p greater than 0.5). Measurements taken later in the procedure documented a significant trend toward baseline in mild and moderate groups. At the opening of the second limb of the graft, the compensated values (CO, RV-EDV, and RV-SV) did not change significantly in the groups with mild and no CAD (p greater than 0.2) but approached significance in the group with moderate CAD (p less than 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)


Thoracoabdominal aortic aneurysms associated with celiac, superior mesenteric, and renal artery occlusive disease: methods and analysis of results in 271 patients. Two hundred seventy-one of 1509 patients who underwent thoracoabdominal aortic repairs had either celiac or superior mesenteric or renal artery occlusive disease. These latter patients were treated by endarterectomy or bypass between June 20, 1960 and Jan. 10, 1991. After 1987, the 30-day survival rate was 93% (79 of 85) compared with 90% (245 of 271) before 1988. Multivariate predictors of death were age, postoperative reoperation for bleeding, and cardiac complications (p less than 0.05). Renal complications (13% dialysis, 35 of 271) were associated with preoperative renal dysfunction, elevated preoperative serum creatinine level, urine clearance time of dye, extent of the aorta replaced, coagulopathy, and paraplegia or paraparesis (p less than 0.05). The incidence of postoperative renal dysfunction was reduced by renal artery endarterectomy (p less than 0.05). On univariate analysis the risk of renal failure was reduced by renal artery perfusion with cold Ringer's lactate solution (p less than 0.05). Gastrointestinal complications (9%, 25 of 271) were associated with a history of peptic ulcer disease on multivariate analysis (p less than 0.05). The Kaplan-Meier 5-year survival rates for patients with and without occlusive disease were 53% and 60%, respectively, and at 10 years 37% and 30%, respectively (p = 0.08). We conclude that endarterectomy or bypass of occlusive visceral disease reduces the risk of renal failure after thoracoabdominal aortic aneurysm repairs, does not decrease early or late survival, and does not increase the risk of gastrointestinal complications.


Acute mesenteric ischemia after cardiopulmonary bypass. Acute mesenteric ischemia is an uncommon but catastrophic event after cardiopulmonary bypass. From 1980 to 1990, 16,951 cardiac procedures requiring cardiopulmonary bypass were performed at Emory University Hospital in Atlanta, Ga. Eighteen patients (0.1%) had acute mesenteric ischemia that resulted in intestinal infarction. Emergency cardiac surgery had been performed in 16 of the 18 patients, and all 18 patients were vasopressor dependent for hemodynamic support after surgery. Diagnostic difficulties resulted in the diagnosis of intestinal infarction an average of 9 1/2 days after cardiopulmonary bypass. Nonocclusive mesenteric arterial ischemia was the determined cause in all cases. Statistically significant risk factors associated with acute mesenteric ischemia after cardiopulmonary bypass surgery included (1) emergency cardiac surgery (p less than 0.0001), (2) the use of an intraaortic balloon pump (p less than 0.0001), (3) failed angioplasty requiring emergency surgery (p = 0.0074), (4) prolonged pump time (p = 0.0093), and (5) advanced age (p = 0.0016). A high index of suspicion for mesenteric ischemia after cardiopulmonary bypass in patients with identified risk factors may decrease the diagnostic delay and lead to an improvement in the 67% mortality rate seen in this series.


Are arteriograms necessary in penetrating zone II neck injuries? The evaluation and management of potential arterial injuries in penetrating neck trauma are controversial. Routine surgical exploration or arteriography can be very expensive and time-consuming and can overburden available resources if used in all patients. We reviewed the records of 4035 patients seen in our trauma center during a 20-month period and identified a total of 110 patients (2.7%) with penetrating wounds to zone II of the neck; 50 were from gunshot wounds, 43 from stab wounds, 7 from shotgun injuries, and 10 from lacerations. In 42 (39%) patients there was no arteriogram or surgery based on location of the wounds or lack of any physical findings. None of these patients later had any evidence of an arterial injury. Forty-five patients (40%) had arteriograms based on proximity or a "soft" sign of vascular injury, which included evidence of significant bleeding or a stable hematoma. A total of 15 injuries to major arteries were identified: 3 common carotid, 5 internal carotid, and 7 vertebral. One patient died during initial resuscitation, and four patients went directly to surgery with no preoperative arteriogram for active bleeding and expanding hematoma (n = 1), an expanding hematoma (n = 2), and a large, stable hematoma (n = 1). Only one patient (of the 110) had a significant major arterial injury requiring surgery that was not predicted by physical findings. Nine arterial injuries were treated nonoperatively: six vertebral, two common carotid intimal flaps, and one small distal internal carotid pseudoaneurysm (diagnosed late). Three additional minor external carotid artery injuries were observed with no adverse sequelae.(ABSTRACT TRUNCATED AT 250 WORDS)


Recognition of arterial injury in elbow dislocation. The clinical presentation of patients with elbow dislocations was reviewed to identify those factors indicating an increased risk for arterial injury. Sixty-two patients were treated for 63 elbow dislocations between January 1981 and July 1991. Eight patients (13%) sustained a concomitant arterial injury involving the brachial (7) and radial (1) arteries. Three clinical findings, absence of a radial pulse, open dislocation, and presence of systemic injuries, were correlated with arterial injury. A palpable radial pulse was absent in six (75%) patients with an arterial injury but in only two (4%) with normal vessels (p less than 0.0001, chi square). Five (33%) open dislocations had an associated arterial injury, whereas three (6%) arterial injuries occurred in closed dislocations (p less than 0.006, chi square). Systemic injury occurred in five dislocations (63%) with arterial injuries and 14 dislocations (25%) without arterial injury (p less than 0.04, chi square). Multivariate analysis showed that absence of a radial pulse was the only factor that significantly predicted arterial injury (p less than 0.0001). Although most elbow dislocations are not associated with arterial injury, absence of a radial pulse or presence of an open dislocation or both should alert the clinician to the increased possibility of an associated vascular injury.


The safety, efficacy, and durability of external carotid endarterectomy. Results of 21 external carotid endarterectomies (ECEAs) in 19 patients with symptoms were reviewed retrospectively. No patients died or had new strokes referable to ECEA within 30 days of surgery. Overall, six (32%) patients died during an average 41 months of follow-up (range 1 to 134 months). Persistent symptoms followed five (71%) of seven ECEAs performed for cerebrovascular insufficiency or transient ischemic attacks. One patient treated urgently for an evolving stroke failed to improve and died after hospital discharge. Another patient with crescendo transient ischemic attacks had a preoperative deficit that did not resolve for 6 months and had a second stroke 2 years later. Another patient had a stroke after a contralateral carotid reconstruction but recovered. In contrast, only two (14%) of 14 ECEAs performed for monocular amaurosis fugax had persistent symptoms after surgery. Durability of 16 ECEAs was evaluated by arteriography or duplex scanning. Of six ECEAs closed primarily, three (50%) occluded, one has 60% restenosis, and only two (33%) had no restenosis (mean follow-up 36 months). Of 10 ECEAs closed by patch angioplasty, none occluded, 2 had 20% restenosis, and eight (80%) had no restenosis (mean follow-up 47 months). Life-table analysis indicated improvement in ECEA patency and durability with patch angioplasty (p = 0.011). From these data, ECEA can be performed with relative safety but is more effective for treatment of monocular amaurosis fugax in patients with a microembolic source at the external carotid origin. Patients with any other indications for ECEA did not benefit consistently from this operation.


Is anticoagulation indicated for asymptomatic postoperative calf vein thrombosis? The purpose of this study was to determine the effect of anticoagulation on the incidence of thrombotic propagation and pulmonary embolism in patients with calf vein thrombosis after total hip or total knee arthroplasty. Patients undergoing arthroplasties had prospective surveillance for postoperative deep vein thrombosis by both bilateral contrast venography and venous duplex scanning. Calf vein thrombosis was documented by venography in 42 patients (50 limbs), including 29 of 253 patients undergoing total hip arthroplasty (11.4%) and 13 of 99 patients undergoing total knee arthroplasty (13%). Of patients on whom follow-up duplex scans were performed, heparin followed by warfarin anticoagulation was used in 11 (13 limbs) and withheld in 21 (25 limbs). Propagation of thrombosis to the popliteal or superficial femoral vein or both was detected by serial duplex scanning in 3 of 13 treated limbs (23%) and 2 of 25 untreated limbs (8%), (p = 0.43). All thrombus propagations were detected within 2 weeks of the operative procedure. There were no pulmonary emboli or deaths. Propagation of asymptomatic calf vein thrombosis after arthroplasty was not influenced by anticoagulation, suggesting that postoperative calf vein thrombosis need not be routinely treated. Serial venous duplex scanning is useful to identify the occasional patient in whom thrombotic propagation requiring anticoagulation develops.


The use of arm vein conduits during infrageniculate arterial bypass. To further examine the use of arm vein for bypass to the popliteal or infrapopliteal arteries, we retrospectively reviewed 43 patients undergoing infrageniculate arterial bypass by use of an arm vein as a conduit. Nine grafts were done to the below-knee popliteal artery and 34 to the infrapopliteal arteries. Six grafts were done by use of a single segment of an arm vein, whereas 37 grafts were composites of either multiple segments of arm vein (n = 19) or segments of saphenous and arm vein (n = 18). Mean follow-up time was 15 1/2 months. Initial (30-day) graft patency and limb salvage were 95%. Primary graft patency by life-table analysis was 67% at 1 year and 49% at 3 years. Follow-up examination detected graft stenosis before occlusion in six patients (all of whom were given anticoagulant medication) and three failing grafts were salvaged. This increased overall 3-year secondary graft patency to 64% and 3-year secondary patency for infrapopliteal bypasses to 66%. Eleven of 12 graft occlusions resulted in major amputations (eight were above the knee, and three were below the knee) so that limb salvage paralleled secondary graft patency (63% at 3 years). Thus arm veins provide an excellent alternative venous conduit for infrageniculate arterial bypass, even when composite venous grafts must be used.


Acute arterial thrombosis in the very young. The case records of all infants under the age of 6 months who underwent surgery for acute arterial thrombosis between January 1980 and September 1991 were reviewed. Seven infants (nine ischemic limbs) were identified and ranged in age from 5 days to 5 1/2 months (mean 2.4 months); all weighed less than 5 kg (mean 3.9 kg). The cause in each case was iatrogenic. Diagnosis was based on the presence of a cool, mottled extremity associated with the absence of insonated peripheral arterial Doppler signals. Treatment included aortoiliac thrombectomy (n = 2), femoral artery thrombectomy with primary closure (n = 4), femoral artery thrombectomy with autogenous saphenous vein patch (n = 1), and axillary artery thrombectomy with end-to-end anastomosis (n = 1). Palpable pulses were restored in five (56%) of nine limbs and Doppler signals in the remaining limbs. There were no instances of limb loss. Excluding aortoiliac thrombectomy, palpable peripheral pulses were reestablished in only 40% of extremities. Thrombectomy is a safe and simple procedure in even the very youngest of patients with arterial insufficiency, but surgical optimism should be tempered by frequent inability to achieve full and durable success.


Noninvasive detection of iliac artery stenosis in the presence of superficial femoral artery obstruction. In patients with superficial femoral artery obstruction, iliac disease may be difficult to diagnose by commonly used noninvasive techniques. We studied common femoral artery acceleration time (onset of systole to peak systole), as measured from a Doppler spectral display and expressed in milliseconds. Previous work has suggested that an acceleration time of 144 msec or greater is abnormal and is associated with iliac stenosis (greater than or equal to 75% diameter reduction) or occlusion. During a 2-year period, in 139 limbs with superficial femoral artery obstruction, acceleration times were measured immediately before angiography. The overall test accuracy was 94.2% (131/139). In the 112 sides with normal angiograms, the acceleration time correctly identified no disease in 109 patients (97.3% specificity), and in the 27 iliac stenoses or occlusions the test detected disease in 22 patients (81.5% sensitivity). This appears to be a good test that can be done with equipment usually available in most vascular laboratories.


Endovascular infrainguinal in situ saphenous vein bypass: a multicenter preliminary report. The ideal operative approach for infrainguinal in situ bypass grafting would render the saphenous vein (SV) valves incompetent while occluding venous tributaries from within the SV: an endovascular in situ SV bypass. Forty-six femoropopliteal-tibial in situ bypasses were performed in part by the endovascular occlusion technique. Valvulotomy was accomplished with a retrograde "cutter" valvulotome, and endoluminal cannulation of 84 SV tributaries was performed with a shape memory metal alloy (nickle-titanium), electronically steerable catheter under angioscopic surveillance. Sixty-nine SV tributaries (82%) were totally occluded and 15 (18%) were partially occluded with platinum occlusion coils. Twelve coils that "recoiled" into the SV lumen were retrieved uneventfully. The valvulotomes caused six SV perforations that were repaired without consequence. Intraoperative fluoroscopy confirmed coil placement and verified venous tributary occlusion, as well as SV graft patency. During short-term follow-up (mean 9.2 months; range 1 to 15 months), all patients have undergone ultrasonography of the in situ bypasses. All 69 of the SV tributaries that occluded initially have remained occluded and 84% (39/46) of the in situ bypasses have remained patent. This study demonstrates that an electronically steerable nitinol catheter can be used safely to occlude venous tributaries from within the SV. Endovascular occlusion of SV tributaries may ultimately obviate the need for long incisions the length of the leg, thus reducing wound-related problems and shortening recuperation.


The effect of unilateral internal carotid arterial occlusion upon contralateral duplex study: criteria for accurate interpretation. To determine the influence of unilateral internal carotid arterial occlusion (ICO) on Doppler frequency spectral analysis (DFSA) of the patent contralateral carotid artery, a retrospective review of 154 patients between July 1987 and December 1991 with angiographically confirmed ICO was performed, correlating duplex and arteriographic findings in a blinded fashion. Biplane arteriograms and bilateral carotid artery duplex studies that used a 5.0 MHz Doppler probe with a 1.5 mm3 sample volume at a 60 degree angle of insonation were performed on all patients. Each carotid artery was categorized by the severity of stenosis as quantified by arteriography: 1% to 15% (n = 41); 16% to 49% (n = 48), 50% to 79% (n = 21), 80% to 99% (n = 34), and bilateral occlusion (n = 10). DFSA peak systolic frequencies were commonly exaggerated in the presence of contralateral ICO and use of standard criteria for DFSA interpretation overestimated bifurcation stenoses in 43 of 89 lesions (48.3%) when determining nonhemodynamically significant lesions (less than 50% diameter reduction) with a sensitivity of only 57.3% and specificity of 96.9%. Conversely, prediction of hemodynamically significant lesions (greater than 50% diameter reduction) with standard criteria had 96.9% sensitivity but only 57.3% specificity. Modification of these criteria to account for the velocity increase or "jet effect" in the ipsilateral carotid artery system increased the sensitivity and specificity to 97.8% in predicting nonhemodynamically and hemodynamically significant stenoses respectively. A Doppler frequency spectrum with a peak systolic frequency (PSF) greater than 4.0 kHz and end-diastolic frequency (EDF) less than 5 kHz with an "open window" distinguished lesions with less than 50% diameter reduction.(ABSTRACT TRUNCATED AT 250 WORDS)


Oesophageal carcinoma in Karachi.

Oesophageal carcinoma in Karachi. Between January, 1979 to August, 1990, 107 histologically proven cases of oesophageal carcinoma were seen. The ages of the patients ranged from 19-85 years (mean 55 +/- 14 years) with a male to female ratio of 1.2:1. Majority (70%) of the cases belonged to lower socioeconomic group and 56% were migrants from India. History of tobacco chewing or smoking was present in 78% cases. In 54%, the lesion was located in the middle third of the oesophagus, followed by lower third in 44%. Histology showed squamous cell carcinoma in 86% and adenocarcinoma in 10% cases. Of the 19 cases followed, 16 underwent surgery and 3 received chemotherapy. Two cases died within 7 months following surgery and in 6 carcinomas recurred. All cases receiving chemotherapy died within 8 months of treatment.


Comparative study of single dose and five days dose of gentamicin as prophylaxis in appendicectomy. One hundred patients who had appendicectomy for uncomplicated acute appendicitis over 1 year were randomly allocated to two groups of 50 patients each. Patients in group 1 had a single prophylactic injection of gentamicin while those in group 2 had a five days course with the first dose given just before the operation. There was no wound infection in any of the patients in either groups. There was also no statistically significant difference in the incidence of other postoperative complications. These findings suggest that a single prophylactic dose of gentamicin was as effective as a five days course in preventing postoperatively complications, like wound infection.


Vulnerability factors for depression in Pakistani women. The vulnerability factors were tested in a retrospective study on 50 cases and 50 controls. Only one of the four factors, i.e., lack of an intimate, confiding relationship, was found to be statistically significant, thus highlighting the need for indigenous research particularly in areas where the variables are socio-culturally determined.


[Fluorescence polarization immunoassay of isepamicin in blood spotted on filter paper]. A simple method has been developed for the determination of the aminoglycoside antibiotic isepamicin (ISP) in whole blood, using dried blood spots (DBSs) on filter-paper. ISP in the DBSs were recovered most effectively in 0.5 M Na2HPO4-NaH2PO4 buffer (pH 7) by incubation for 30 minutes at 35 degrees C in an ultrafiltration tube. The eluates from the DBS papers were centrifuged at 3,000 x g for 10 minutes. The clear, colorless filtrates were transferred to an Abbott TDx cartridge for measurement by the fluorescence polarization immunoassay. The lower limit for the quantitative determination of ISP in the DBS was 2.5 micrograms per ml of whole blood. The method permits a simple collection of blood at a microliter level and should prove particularly useful for therapeutic drug monitoring of ISP in blood at effective levels in pediatric and geriatric patients.


[In vitro study on efficacy of combination use of aspoxicillin and beta-lactam preparations (ceftazidime, cefmetazole and aztreonam) against bacteria isolated from abdominal infections]. An in vitro study was done to evaluate combination use of aspoxicillin (ASPC) with each of 3 beta-lactam preparations, ceftazidime (CAZ), cefmetazole (CMZ) and aztreonam (AZT). The results obtained are summarized as follows: 1. ASPC has strong activity against Gram-positive bacteria and anaerobic bacteria, while CMZ and CAZ have strong activity against Gram-negative bacteria. 2. Rates of beta-lactamase producing strains among the isolated bacteria (a total of 383 isolates) were 4.4% among Gram-positive bacteria, 71.6% among Gram-negative bacteria and 89.3% among anaerobic bacteria. The overall rate of beta-lactamase secreting strains among all isolates was 46.5%. 3. Efficacies of combination uses were studied using the FIC index. Combination of ASPC and CAZ was effective against 95.0% of the isolates, ASPC and AZT against 85.7%, and ASPC and CMZ against 83.5%. Combination of ASPC and CMZ showed antagonism in 12.8% of the isolates. In conclusion, combination use of ASPC with any one of CMZ, AZT or CAZ proved to be highly effective. In particular, combination of ASPC and CAZ appeared to be the best in view of complementing antibacterial spectra.


[Antimicrobial activities of ceftriaxone against clinically isolated strains]. Antibiotic activities (MICs) of ceftriaxone (CTRX) against 1,210 strains of bacteria including 28 spp. isolated in 1987 and 1990 were compared with those of other cephems. 1. When compared to data on clinically isolated strains reported in the early 1980s, strains of the following species isolated in 1990 showed extremely elevated MIC90s of CTRX: Staphylococcus spp., Streptococcus pneumoniae, Escherichia coli, Citrobacter spp., Enterobacter spp., Serratia spp., Proteus vulgaris, Morganella morganii and Providencia spp. No changes were observed in MIC90s between the 2 periods for microorganisms such as Streptococcus pyogenes, Haemophilus influenzae, Klebsiella pneumoniae, Proteus mirabilis and Peptostreptococcus spp. 2. The MIC90 of CTRX to S. pneumoniae was high because a large number of benzylpenicillin (PCG)-insensitive S. pneumoniae (PISP) was present among this species. The MIC80 to Bacteroides fragilis group was also high because highly resistant B. fragilis and B. thetaiotaomicron were isolated in large proportions among the bacteria of this group. Other oxime-type cephems also had high MICs against the above mentioned bacteria. Therefore, a further evaluation has to be made with regard to activities of oxime-type cephems such as CTRX against PISP and B. fragilis group. 3. Sample strains included, in high ratios, methicillin-resistant Staphylococcus aureus (MRSA), cephamycin-resistant as well as oxime-type cephem-resistant intestinal bacteria, Gram-negative bacteria, and new-quinolone-resistant bacteria. Some of there resistant bacteria are also CTRX-resistant, and CTRX had insufficient activities against them. 4. With regard to the assessment of changes of frequencies of specific drug-resistant bacteria, including those with CTRX-resistance from year to year, the authors would like to point out the following comment of theirs made in 1989 and 1991, which appears to be increasing its significance, "Subjects of future studies should include dose on the mechanisms for the acquisition of bacterial resistance to entire beta-lactam antibiotics and the social circumstances in which resistant bacteria appear". 5. It appears that those strains resistant to cephems including CTRX are increasingly found among clinically isolated strains in recent years. CTRX, however, was found still effective against most clinical pathogens. Furthermore, considering that CTRX is one of the few drugs which sustain high blood concentrations of active forms we concluded that CTRX is a useful cephem-group antibiotic.


[Clinical evaluation of combined therapy of ceftazidime and tobramycin for intractable pulmonary infection mainly caused by Pseudomonas aeruginosa]. In an open, multicenter trial, we investigated the clinical efficacy of a combination therapy of ceftazidime (CAZ) and tobramycin (TOB) for intractable pulmonary infections mainly caused by Pseudomonas aeruginosa. Evaluated for the utility of the combination therapy were 33 cases with pneumonia (Group I: pneumonia caused by P. aeruginosa 15, Group II: pneumonia caused by other Gram-negative bacilli 4 and pneumonia which causative organism was not determined 14) and 23 cases with chronic respiratory tract infection caused by P. aeruginosa. The results obtained are summarized as follows. 1. In Group I pneumonia, included 11 severe cases and 4 moderate cases, with a mean age of 69.3 years. Significant underlying diseases were present in 14 out of the 15 (93.3%): they included 10 cases of pulmonary diseases and 4 cerebrovascular diseases. The overall efficacy rate in these cases was 60.0%: but the efficacy rate in moderate cases was 100% and that in severe cases was 45.5%. 2. In Group II pneumonia included 16 severe cases and 2 moderate cases with a mean age of 68.2 years. Significant underlying diseases were present in 15 out of 18 (83.3%, all of the underlying diseases were pulmonary diseases) and the overall efficacy rate was 72.2% with 100% efficacy rate among moderate cases and 68.8% among severe cases. 3. In the cases with chronic respiratory tract infections caused by P. aeruginosa, the efficacy rate was 82.6% and the eradication rate was 65.2%. We consider the combination therapy of CAZ and TOB is useful for intractable pulmonary infections caused by P. aeruginosa.


[Transferability of meropenem to cerebrospinal fluid in rabbits with meningitis caused by Staphylococcus aureus]. The transferability of meropenem (MEPM) to cerebrospinal fluid (CSF) was studied employing rabbits with experimental meningitis caused by Staphylococcus aureus. The mean serum concentration was 93.1 +/- 13.5 micrograms/ml at 15 minutes after intravenous administration of MEPM at a dose level of 100 mg/kg. The mean concentration in CSF was maximum at 15 minutes after administration at 4.42 +/- 2.24 micrograms/ml. Pharmacokinetic parameters calculated from these values were as follows: Cmax (CSF/serum) 4.75%, AUC (CSF/serum) 10.4% between 15 and 60 minutes, 13.9% between 15 and 120 minutes and 15.7% between 15 and 180 minutes, T 1/2 for MEPM in CSF: 50.9 minutes, T 1/2 (CSF/serum): 2.19. In comparison to those of imipenem which were obtained in the same way, the transferability of MEPM was similar and in consideration of the antimicrobial potency against the main pathogens of meningitis, it appears worth-while of running clinical trials for this drug.


[Clinical and pharmacokinetic evaluations of meropenem in children]. Twenty-five children were treated with meropenem (MEPM, SM-7338) and the clinical efficacy and side effects were evaluated. Ages of the patients ranged from 9 months to 11 years. Dose levels of MEPM ranged from 50.4 to 108 mg/kg/day for 4 to 8 days. The 25 patients included 11 pneumonia cases, 4 bronchitis, 6 tonsillitis, 3 urinary tract infections and 1 gingivitis, and they were evaluated for the clinical efficacy of MEPM. Results were excellent in 13 and good in 12 patients. No side effects nor abnormal clinical laboratory test results were observed. The pharmacokinetics of MEPM was studied in 7 patients with ages ranging from 9 to 15 years. The mean plasma peak concentration of MEPM in 5 patients was 36.7 micrograms/ml after dosing 10 mg/kg, and that of 2 patients was 70.0 micrograms/ml after administering 20 mg/kg. These data showed that plasma concentrations of drug depended on dose levels. Average half-life values for the 2 groups (10 and 20 mg/kg) were 0.83 and 0.85 hour, respectively. Urinary recovery rates for the 2 groups (10 and 20 mg/kg) were 64.3% and 81.3%, respectively, in the first 5 hours after administration.


[Clinical evaluation of meropenem in the pediatric field]. A clinical study on a new carbapenem antibiotic, meropenem (MEPM), was carried out in acute pediatric infections. MEPM was administered to 8 patients including 3 patients with acute pneumonia, 2 with cervical lymphadenitis, 1 with acute tonsillitis, and 1 with cellulitis and 1 with sepsis. The overall efficacy rate was 100%. As an adverse reaction, diarrhea was observed in 1 patient. In clinical laboratory tests 1 patient was found to have S-GPT elevation which normalized after discontinuation of MEPM. MEPM appears to be effective and safe drug for pediatric acute infections.


[Pharmacokinetic, bacteriological and clinical studies on meropenem in children]. Pharmacokinetic, bacteriological and clinical studies on meropenem (MEPM) were performed in children. The results are summarized as follows: 1. A total of 16 patients was treated with MEPM. Each dose was 20 mg/kg, and administration was made 3 times daily using 30-minute intravenous drip infusion for 5-28 days. Clinical efficacies of MEPM in 16 patients with bacterial infections (1 with purulent meningitis, 1 with suspected subdural abscess, 2 with suspected sepsis, 4 with pneumonia, 1 with acute maxillar sinusitis, 2 with cervical abscess, 1 with acute gastroenteritis, 2 with skin soft tissue infection and 2 with urinary tract infection) were evaluated as excellent in 7 patients, good in 8 patients and fair in 1 patient with an efficacy rate of 93.8%. Fourteen causative organisms found in 11 patients (Streptococcus pneumoniae in 4, Branhamella catarrhalis in 3, Staphylococcus aureus in 3, Group B Streptococcus in 1, Escherichia coli in 3) were all eradicated. No adverse reactions were observed in any of the 16 patients. 2. MICs of MEPM against 6 clinically isolated bacteria (B. catarrhalis 2, S. pneumoniae 3 and S. aureus 1) from children with bacterial infections were examined. MEPM showed good antibacterial activities. 3. Pharmacokinetic studies: Peak plasma concentrations of MEPM averaged 43.07 micrograms/ml (37.20-46.30 micrograms/ml) at dose of 20 mg/kg administered by 30-minute drip infusion. In the first 8 hours after administration, the urinary excretion rates of MEPM averaged 39.9% of the administered dose.(ABSTRACT TRUNCATED AT 250 WORDS)


[Studies of meropenem in pediatric infections]. Pharmacokinetic and clinical evaluations of meropenem (SM-7338, MEPM) were carried out in pediatric patients. The following results were obtained. 1. After 30-minute intravenous drip infusion at a dose of 20 mg/kg, plasma concentrations of MEPM reached their peaks at the end of drip infusion with an average value of 48.8 +/- 3.64 micrograms/ml, and the average plasma half-life was 0.93 +/- 0.21 hour in the beta-phase. After 30-minute intravenous drip infusion at a dose of 10 mg/kg, the average peak plasma concentration was 27.7 +/- 4.33 micrograms/ml and the average plasma half-life was 0.78 +/- 0.20 hour. 2. Urinary excretion rates of MEPM after 30-minute intravenous drip infusion at doses of 20 and 10 mg/kg were 44.8 +/- 4.54% and 40.9 +/- 1.78%, respectively. 3. MEPM was administered to 13 cases (upper and lower respiratory infections, pneumonia and lymphadenitis) at daily doses between 60-90 mg/kg/day divided into 3 dosages using 30-minute intravenous drip infusion. Clinical responses were "excellent" in 12 patients, "good" in 1, hence an efficacy rate of 100% was obtained. 4. Bacteria identified in various disease cases included 12 strains of 5 species, and the eradication rate was 100%. 5. No side effects were observed in any children. Laboratory test results showed abnormalities in 2 cases with elevations of GOT and GPT. These results suggest that MEPM may be a very useful and safe drug for the treatment of pediatric infections.