Biomedical subjects
Publications and source records attributed to J R Brown.
A commercial inline constant flow valve for hand-compression hydraulic sprayers was tested to evaluate its capability to maintain constant pressure to the nozzle down to a preset cut-out pressure. The flow-control valve provided consistent flow rates when used with the H.D. Hudson X-Pert 9.5-liter hand-compression sprayers. This flow valve may have potential for use in vector control operations.
This study explores the potential of the monoglyceride monocaprin as an enhancer of the epithelial permeability of the beta(3)-adrenoceptor agonist BTA-243, as an approach to improving the bioavailability of this drug. The permeabilities of BTA-243 and mannitol (paracellular marker) in Caco-2 cell monolayer and everted gut sac models in aqueous buffer (pH 6.8) in the presence of 1.3 and 2.0 mM monocaprin were compared with control (monocaprin-free) solutions over a period of 1 h. The transepithelial electrical resistance (TEER) of the Caco-2 cell monolayers was measured at regular time intervals throughout the experiment and after a recovery period of 30 h. Toxicological damage to the biological models associated with exposure to monocaprin was assessed by scanning electron microscopy and by the measurement of lactate dehydrogenase (LDH) release from everted gut sacs. The permeability of BTA-243 in epithelial monolayers was enhanced in the presence of 1.3 and 2.0 mM monocaprin. Measurements of TEER and mannitol permeability showed partial recovery of barrier properties after a 30 h period following exposure to 1.3 mM monocaprin. No structural damage was evident in these monolayers. Enhancement of Caco-2 permeability to BTA-243 by 2.0 mM monocaprin was significantly greater than by 1.3 mM but was irreversible; monolayers failed to recover their barrier properties after 30 h and changes in their gross morphology were observed. The mucosal to serosal transfer of BTA-243 in everted gut sac was enhanced but to a lesser extent than in the Caco-2 model. LDH release from everted gut sacs exposed to monocaprin was significantly less than that after exposure to Triton X-100, a nonionic surfactant known to cause membrane disruption.
Four handheld aerosol-mist generators and 1 thermal fog generator were evaluated initially for their ability to deliver low flow rates (< or = 5 ml/min) of resmethrin insecticide. Two generators, the London Fog Eliminator and Clarke Pl, were then selected from that group to conduct a treatment of a block of 16 residences (4 x 4 grid) vs. direct treatment of individual residences to determine the most efficacious method of treating a group of small dwellings or a village. Data are presented on droplet density, volume median diameter, and mortality of caged mosquitoes treated with the P1 and Eliminator in residences treated as a group; and mortality for caged Aedes aegypti L. in residences treated individually with the P1 at various flow control settings and spray-on times.
A family of five beta1,3-galactosyltransferases has been characterized that catalyze the formation of Galbeta1,3GlcNAcbeta and Galbeta1,3GalNAcbeta linkages present in glycoproteins and glycolipids (beta3GalT1, -2, -3, -4, and -5). We now report a new member of the family (beta3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, beta3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the beta3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine- or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal beta-linked galactose residue. This product, Galbeta1,3Galbeta is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcAbeta1,3Galbeta1,3Galbeta1,4Xylbeta-O-Ser), indicating that beta3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells. Its localization in the medial Golgi indicates that this is the major site for assembly of the linkage region.
A Deltalgt (Lgt, lipoprotein diacylglyceryl transferase) isogenic mutant was obtained which indicates that lgt is not essential for cell growth in vitro, like in the Gram-positive bacterium Bacillus subtilis, but unlike in the proteobacteria Escherichia coli and Salmonella typhimurium. The mutation was transduced to a virulent strain. A 5 log attenuation was observed in a respiratory tract model of infection. Metabolic labeling by [U-14C]palmitate revealed the presence of eight to ten lipoproteins in the wild-type strain only, with molecular masses between 15 and 80 kDa. Our findings suggest a major difference in the role of lipoproteins in Gram-positive bacteria versus the proteobacteria.
Horizontal gene transfer (HGT) has long been recognized as a principal force in the evolution of genomes. Genome sequences of Archaea and Bacteria have revealed the existence of genes whose similarity to loci in distantly related organisms is explained most parsimoniously by HGT events. In most multicellular organisms, such genetic fixation can occur only in the germ line. Therefore, it is notable that the publication of the human genome reports 113 incidents of direct HGT between bacteria and vertebrates, without any apparent occurrence in evolutionary intermediates, that is, non-vertebrate eukaryotes. Phylogenetic analysis arguably provides the most objective approach for determining the occurrence and directionality of HGT. Here we report a phylogenetic analysis of 28 proposed HGT genes, whose presence in the human genome had been confirmed by polymerase chain reaction (PCR). The results indicate that most putative HGT genes are present in more anciently derived eukaryotes (many such sequences available in non-vertebrate EST databases) and can be explained in terms of descent through common ancestry. They are, therefore, unlikely to be examples of direct HGT from bacteria to vertebrates.
This investigation has examined possible causes of the poor bioavailability of the beta(3)-adrenoceptor agonist BTA-243. The aqueous solubility of BTA-243 is pH dependent with a solubility minimum at pH 1.5. However, the dissolution rate of the disodium salt of BTA-243 is similar at both pH 2.0 and 7.4 indicating that dissolution rate is unlikely to be the controlling factor in the absorption of BTA-243. The apparent permeability coefficient of BTA-243 across Caco-2 monolayers at pH 6 was lower than that of mannitol and therefore the epithelial permeability of the molecule in vivo is predicted to be very low and potentially bioavailability limiting. Apparent permeability coefficients were not dependent on BTA-243 concentration over the concentration range 0.5 to 12 mM, indicating that epithelial transport is unlikely to occur via a saturable mechanism. They were of similar magnitude in both directions across the monolayers, indicative of no significant effluxing of BTA-243 by components of the cell membrane. Apparent octanol/water distribution coefficients increased with decrease of pH between 2 and 6; the relatively low values at pH 4 and 6 suggest that the limited intestinal absorption predicted in vivo will occur predominantly via paracellular passive diffusion. Everted gut sac experiments performed at pH 2.0 and 6.8 suggest that at pH 2.0 a significant proportion of the BTA-243 transport occurs via the transcellular route confirming that the ionization state of the BTA-243 molecule influences the route and rate of epithelial permeability.
Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.
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A series of synthetic mannosides was screened in a cell-free system for their ability to act as acceptor substrates for mycobacterial mannosyltransferases. Evaluation of these compounds demonstrated the incorporation of [14C]Man from GDP-[14C]Man into a radiolabeled organic-soluble fraction and analysis by thin layer chromatography and autoradiography revealed the formation of two radiolabeled products. Each synthetic acceptor was capable of accepting one or two mannose residues, resulting in a major and a minor mannosylated product. Both products from each acceptor were isolated and their mass was confirmed by fast-atom bombardment-mass spectrometry (FABMS). Characterization of each mannosylated product by exo-glycosidase digestion. acetolysis and linkage analysis by gas chromatography mass spectrometry of partially per-O-methylated alditols, revealed only alpha1-6-linked products. In addition. the antibiotic amphomycin selectively inhibited the formation of mannosylated products suggesting polyprenolmonophosphate-mannose (C15 50-P-Man) was the immediate mannose donor in all mannosylation reactions observed. The ability of synthetic disaccharides to act as acceptor substrates in this system, is most likely due to the action of a mycobacterial polyprenol-P-Man:mannan alpha1-6 mannosyltransferase involved in the biosynthesis of linear alpha1-6-linked lipomannan.
Universal trees of life based on small-subunit (SSU) ribosomal RNA (rRNA) support the separate mono/holophyly of the domains Archaea (archaebacteria), Bacteria (eubacteria) and Eucarya (eukaryotes) and the placement of extreme thermophiles at the base of the Bacteria. The concept of universal tree reconstruction recently has been upset by protein trees that show intermixing of species from different domains. Such tree topologies have been attributed to either extensive horizontal gene transfer or degradation of phylogenetic signals because of saturation for amino acid substitutions. Here we use large combined alignments of 23 orthologous proteins conserved across 45 species from all domains to construct highly robust universal trees. Although individual protein trees are variable in their support of domain integrity, trees based on combined protein data sets strongly support separate monophyletic domains. Within the Bacteria, we placed spirochaetes as the earliest derived bacterial group. However, elimination from the combined protein alignment of nine protein data sets, which were likely candidates for horizontal gene transfer, resulted in trees showing thermophiles as the earliest evolved bacterial lineage. Thus, combined protein universal trees are highly congruent with SSU rRNA trees in their strong support for the separate monophyly of domains as well as the early evolution of thermophilic Bacteria.
Prokaryotes have been at the forefront of the genome sequencing revolution. Many genomes have been completely sequenced, revealing much about bacterial and archaeal genome content and organization. Yet, a meaningful evolutionary picture of prokaryotes still eludes us. Much of the problem lies in understanding the mode and tempo of genome evolution. Here phenylalanyl-tRNA synthetase is used as an example of the complex interplay among lateral gene transfer, operon recombination, and gene recruitment in the evolution of some prokaryotic genes. Promising new approaches to genomic analyses, which could add to our understanding prokaryotic evolution and help in their classification, are discussed.
We report the characterization of two Chinese hamster ovary cell lines that produce large amounts of sulfated N-linked oligosaccharides. Clones 26 and 489 were derived by stable transfection of the glycosaminoglycan-deficient cell mutant pgsA-745 with a cDNA library prepared from wild-type cells. Peptide:N-glycanase F released nearly all of the sulfate label, indicating that sulfation had occurred selectively on the Asn-linked glycans. Hydrazinolysis followed by nitrous acid treatment at pH 4 and borohydride reduction yielded reduced sulfated disaccharides that comigrated with standard Gal3SO4beta1-4anhydromannitol. The disaccharides were resistant to periodate oxidation but became sensitive after the sulfate group was removed by methanolysis, indicating that the sulfate was located at C3 of the galactose residues. Maackia amurensis lectin bound to the sulfated glycopeptides on the cell surface and in free form, even after sialidase treatment. This finding indicates that the lectin requires only a charged group at C3 of the galactose unit and not an intact sialic acid. Growth of cells with chlorate restored sialidase sensitivity to lectin binding, indicating that sulfation and sialylation occurred largely at the same sites. The enhanced sulfation was due to elevated sulfotransferase activity that catalyzed transfer of sulfate from phosphoadenosine-5'-phosphosulfate to Galbeta1-4(3)GlcNAcbeta-O-naphthalenemethanol.
A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.
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Although heparin is widely used as an antithrombotic agent, its multiple mechanisms of action are not fully defined. Recent work has suggested that tissue factor pathway inhibitor (TFPI) may contribute to the antithrombotic activity of heparin by inhibiting the extrinsic pathway of coagulation. We have investigated the effect of heparin on TFPI and have found that when unfractionated heparin is given by continuous intravenous infusion to hospitalized inpatients, TFPI levels increase 2.3-fold and remain high as long as heparin is continued, but return to baseline levels soon after the infusion is stopped. In contrast, therapeutic doses of the low molecular weight heparin, dalteparin, resulted in significantly less TFPI induction. Given the increasing number of studies establishing the clinical efficacy of low molecular weight heparins as antithrombotic agents, these results suggest that TFPI may not be a major contributor to the antithrombotic effect of heparin.
Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-( 1 --> 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranoside (6), 2-naphthylmethyl 2-aceta-mido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2,4,6-tri-O-acetyl-beta-D-galactopyranoside (14), 2-naph-thylmethyl2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1 --> 3)-2-acetamido-4,6-di- O-acetyl-2-deoxy-alpha-D-galactopyranoside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glu-copyranosyl-(1 --> 6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (27). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin.
The origin of the eukaryotic cell remains an unsolved question. Numerous experimental and phylogenetic observations support the symbiotic origin of the modern eukaryotic cell, with its nucleus and (typically) mitochondria. Incorporation of mitochondria has been proposed to precede development of the nucleus, but it is still unclear whether mitochondria were initially part of basal eukaryotes. Data on alanyl-tRNA synthetase from an early eukaryote and other sources are presented and analyzed here. These data are consistent with the notion that mitochondrial genesis did not significantly precede nucleus formation. Moreover, the data raise the possibility that diplomonads are primary amitochondriates that radiated from the eukaryotic lineage before mitochondria became fully integrated as a cellular organelle.