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Biomedical subjects

R L Hill

Publications and source records attributed to R L Hill.

At least 19 recordsLinked to original sources
PubMed2005

Carbaryl, 2,4-D, and Triclopyr adsorption in thatch-soil ecosystems.

Thatch development in intensively managed turf sites may cause environmental concerns for greater sorption or leaching of applied chemicals in terrestrial ecosystems. To determine the adsorption potential of Carbaryl (1-Napthyl N-methylcarbamate), 2,4-D (2,4-dichloro-phenoxyacetic acid), and Triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) in turf ecosystems, composite thatch and underlying soil samples from three-and six-year-old stands of cool-season Southshore creeping bentgrass (Agrostis palustris Huds.) and warm-season Meyer zoysiagrass (Zoysia japonica Steud.) were collected. The samples were processed and analyzed for total organic carbon (COrg); extractable (CExt), humic (CHA) and fulvic acid (CFA); anthrone reactive nonhumic carbon (ARC) fractions; and CHA and CFA associated iron (Fe) contents. Pesticide adsorption capacity (Kf) and intensity (1/n), organic carbon partition coefficient (KOC) and Gibbs free energy change (deltaG) were calculated for thatch materials and the underlying soils using a modified batch/flow technique. Both bentgrass (BT) and zoysiagrass thatch (ZT) contained a greater concentration of CExt, CFA, CHA, and ARC than the respective soils (BS and ZS). The CExt, CFA, CHA, and ARC concentration was higher in BT compared with ZT. The BT contained a greater concentration of bound Fe in both CFA and CHA fractions than in BS, whereas ZT had more bound Fe in CHA fraction than in ZS. On average, the BT had a greater concentration of bound Fe in CExt, CFA, and CHA fractions than in the ZT. Among the pesticides, Carbaryl had higher Kf and 1/n values than 2,4-D and Triclopyr for both thatch and soil. Although the KOC and deltaG values of Carbaryl were higher in both BT and ZT than in the underlying soils, the KOC and deltaG values of 2,4-D were significantly higher in BS and ZS than in the overlying thatch materials. The 2,4-D and Triclopyr had higher leaching indices (LI) than Carbaryl for both BT and ZT materials than the respective soils. The Carbaryl, however, had a higher LI for soils than for thatch materials. Averaged across thatch materials and soils, COrg accounted for 96, 85, and 84% variations in Carbaryl, 2,4-D, and Triclopyr adsorption, respectively. Among the COrg fractions, lignin followed by CFA and CHA accounted for greater adsorption of pesticides, especially Carbaryl. The concentration of CHA and CFA bound Fe did not correlate with Kf and 1/n values of pesticides.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Effect of mutations in Gag on assembly of immature human immunodeficiency virus type 1 capsids in a cell-free system.

Studies of HIV-1 capsid formation in a cell-free system revealed that capsid assembly occurs via an ordered series of assembly intermediates and requires host machinery. Here we use this system to examine 12 mutations in HIV-1 Gag that others studied previously in intact cells. With respect to capsid formation, these mutations generally produced the same phenotype in the cell-free system as in cells, indicating the cell-free system's high degree of fidelity. Analysis of assembly intermediates reveals that a mutation in the distal region of CA (322 LDeltaS) and truncations proximal to the second cys-his box in NC block multimerization of Gag at early stages in the cell-free capsid assembly pathway. In contrast, mutations in the region of amino acids 56-68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations distal to the first cys-his box in NC and mutations in the distal half of MA (88HDeltaG, 85YDeltaG, Delta104-115, and Delta115-129), do not affect formation of immature capsids in the cell-free system. These data provide new information on the role of different domains in Gag during the early events of capsid assembly.

Amino Acid Sequence↗
PubMed2001

Recent advances in the chemistry of spinosyns.

The spinosyns are a new class of fermentation-derived insect control agents that are effective against a variety of chewing insect pests. The successful introduction of spinosad into the agricultural marketplace represents an important milestone in the use of natural products for commercial pest control. The development of a natural product presents additional limitations relative to a synthetic material. While the latter affords some degree of control in building appropriate physical attributes such as photostability, a natural product, designed to function in a different environment, is often less suited for traditional spray applications. Despite its intrinsic photolability, spinosad is stable enough to perform under field conditions. In an effort to generate analogs with improved physical characteristics, we have developed a variety of conditions for selectively modifying different portions of the molecule, and we have discovered analogs with greater activity against a broader spectrum of pests. The inability to translate improved greenhouse activity to actual field conditions resulted in a detailed study of the effects of formulations and crystallinity on biological activity. Through this effort, measurably improved field performance of synthetic spinosyn analogs relative to the natural product have now been observed.

Animals↗
PubMed2001

Intra- and inter-generic plasmid-mediated spread of cephalosporin and aminoglycoside resistance amongst Klebsiella aerogenes K41 and other enterobacteria.

Klebsiella aerogenes K41, resistant to third generation cephalosporins and aminoglycosides, was isolated from clinical samples of 153 in-patients. Blood cultures accounted for 24 (15.7%) of isolates. The MIC(90) of ceftazidime for the isolates of 84 patients was >512 mg/l and was reduced to 2.0 by 4 mg/l of clavulanic acid, but only to 64 by 4 mg/l of sulbactam. Isolates of K. aerogenes K41 produced extended-spectrum beta-lactamase (ESBL) SHV-5 and TEM-1, identified by isoelectric focusing. Plasmid profiles showed that co-dissemination of cephalosporin and aminoglycoside resistance, plus ESBL production, coincided with the acquisition of a 116-kb plasmid. This plasmid was transferable in vitro from K. aerogenes K41 to other serotypes and genera of the Enterobacteriaceae.

Aminoglycosides↗
PubMed2000

The in-vitro activity of povidone-iodinecream against Staphylococcus aureus and its bioavailability in nasal secretions.

Due to the emergence of mupirocin-resistance in some epidemic strains of methicillin resistant Staphylococcus aureus (EMRSA) and the appearance of EMRSA with intermediate resistance to vancomycin, we evaluated the in-vitro activity of 5% povidone-iodine ('Betadine') cream as a possiblealternative to mupirocin for the elimination of nasal carriage of S. aureus. As judged by enrichment culture, povidone-iodine was bactericidal against three mupirocin-sensitive strains of S. aureus from nasal carriers, and against mupirocin-resistant and -sensitive strains of EMRSA types 3, 15 and 16, after incubation with povidone-iodine for 1.0 min at 32 degrees C. Mupirocin nasal ointment did not prevent growth after 180 min incubation. In a quantitative suspension test, 1:100 dilution of povidone-iodine cream completely eliminated an inoculum of 10(8)cfu/mL of all nine test organisms after incubation at 32 degrees C for 1.0 min, and 1:1000 dilution reduced cfu, by a factor of 10(5). After direct inoculation of the povidone-iodine cream to give 10(5)cfu/g, none of the test strains were recoverable after 30 s, giving a killing rate of approximately 10(4)cfu/s; for mupirocin nasal ointment, the maximum reduction of mupirocin-sensitive strains was ten fold after 3 h. Povidone-iodine activity was not detectable in sensitivity-testing agar, although 0.025% of povidone-iodine was detectable in a 15% nutrient strength tryptone soya agar. Using this minimal medium, the addition of nasal secretions (from any of 11 samples) reduced the activity of povidone-iodine by 80-90%, but mupirocin activity was unaffected. One millilitre of nasal secretions inactivated the equivalent of approximately 22.5 mg of povidone-iodine. These results suggest that povidone-iodine cream may have a role in the prevention of colonization and infection caused by MRSA, including mupirocin-resistant strains.

Anti-Bacterial Agents↗
PubMed2000

Infection of foot ulcers with Staphylococcus aureus associated with increased mortality in diabetic patients.

Diabetic patients with foot ulceration have a poorer prognosis than those without ulceration. The reason for this is unclear, but there is considerable interest in the putative links between infection and atherogenesis, and it is notable that diabetic foot ulcers (DFU) are often infected with Staphylococcus aureus and the main cause of death in DFU patients is ischaemic heart disease. We examined the 5 year survival of 71 diabetic patients who presented with foot ulcers that were newly infected (Sa group, n = 56) or not infected at all during the study period (non-Sa group, n = 15) with S. aureus. Twenty-nine patients (52%) infected with S. aureus died compared with three patients (20%) whose foot ulcers were not infected with S. aureus. The patients in the two groups were similar in age and duration of diabetes. The overall five year mortality rate was 10.4% per year for those infected, significantly higher than the average of 4.0% for patients without infection (p = 0.015). None of the patients was bacteraemic or died directly from sepsis. Infection of DFU by S. aureus may increase the risk of death in diabetic patients.

Aged↗
PubMed1998

Identification of the half-cystine residues in porcine submaxillary mucin critical for multimerization through the D-domains. Roles of the CGLCG motif in the D1- and D3-domains.

Plasmids encoding the amino-terminal region of porcine submaxillary mucin were modified by site-specific mutagenesis to assess the roles of individual half-cystine residues in the assembly of disulfide-linked multimers of mucin. COS-7 cells with the plasmid containing C1199A expressed primarily monomers, suggesting that half-cystine 1199 in the D3-domain is involved in forming mucin multimers. This residue is in the sequence C1199SWRYEPCG, which is highly conserved in the D3-domain of other secreted mucins and human prepro-von Willebrand factor. In contrast, cells with the plasmid containing C1276A expressed trimers like those with unmutated plasmid, suggesting that half-cystine 1276 is not involved in formation of disulfide-bonded multimers. The roles of the half-cystines in the CGLCG motifs in the assembly of disulfide-bonded multimers of mucin were also assessed. Cells with plasmids in which both half-cystines in the motif in the D1- or D3-domain of mucin are replaced by alanine expressed proteins that were poorly secreted, suggesting that these mutations impair normal folding of the expressed proteins. A plasmid with a mutant D1-domain motif expressed monomers, whereas one with a mutant D3-domain motif expressed monomers and trimers. However, the trimers expressed by the latter plasmid were assembled in non-acidic compartments, as judged by expression studies in the presence of monensin, which inhibits trimer formation by unmutated plasmid, but not by the mutant plasmid. These results suggest that the CGLCG motif in the D1-domain is required for multimerization in the trans-Golgi complex. However, the CGLCG motif in the D3-domain appears to prevent formation of mucin multimers in non-acidic compartments of the cell. Plasmids encoding the D1- and D2-domains, the D1- and D3-domains, or only the D3-domain also expressed oligomers in the presence of monensin, suggesting that the three D-domains must be contiguous to avoid multimerization in non-acidic compartments. It is possible that these motifs in mucins are engaged in the thiol-disulfide interchange reactions during the assembly of disulfide-bonded multimers of mucin.

Amino Acid Sequence↗
PubMed1998

Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains.

COS-7 cells expressing 1,360 residues from the amino terminus of porcine submaxillary mucin were used to determine whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel electrophoresis showed a single N-glycosylated protein with no indication of proteolytically processed forms. Without prior reduction, only two proteins, corresponding to monomeric and disulfide-linked trimeric species, were observed. The expressed protein devoid of N-linked oligosaccharides also formed trimers, but was secreted from cells in significantly less amounts than glycosylated trimers. Pulse-chase studies showed that the disulfide-linked trimers were assembled inside the cells no earlier than 30 min after protein synthesis commenced and after the intracellular precursors were N-glycosylated. Trimer formation was inhibited in cells treated with brefeldin A, monensin, chloroquine, or bafilomycin A1, although only brefeldin A prevented the secretion of the protein. These results suggest that trimerization takes place in compartments of the Golgi complex in which the vacuolar H+-ATPase maintains an acidic pH. Coexpression in the same cells of the amino-terminal region and the disulfide-rich carboxyl-terminal domain of the mucin showed that these structures were not disulfide-linked with one another. Cells expressing a DNA construct encoding a fusion protein between the amino- and carboxyl-terminal regions of the mucin secreted disulfide-linked dimeric and high molecular weight multimeric species of the recombinant mucin. The presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region of mucin to aid in the assembly of multimers is consistent with its amino acid identities to the amino-terminal region of human von Willebrand factor, which also serves to form disulfide-linked multimers of this protein.

Animals↗
PubMed1998

The carboxyl-terminal 90 residues of porcine submaxillary mucin are sufficient for forming disulfide-bonded dimers.

COS-7 cells transfected with expression vectors encoding 90 and 154 amino acid residues, respectively, from the carboxyl terminus of the disulfide-rich domain (240 residues) of porcine submaxillary mucin were shown to form disulfide-bonded dimers. Cells with expression vectors that encoded the disulfide-rich domain lacking the last 90 and 150 carboxyl-terminal residues, respectively, from the carboxyl terminus of the disulfide-rich domain were unable to secrete truncated domains. These results indicate that the information required to form disulfide-bonded dimers resides in only 90 residues, including 11 half-cystines. Site-specific mutagenesis was employed to change, one at a time, each codon for the 11 half-cystines to serine. Eight of the 11 mutants formed disulfide-bonded dimers indistinguishable from those produced by unmutated vector, although 6 of the 8 mutants also produced aggregates thought to be misfolded protein with scrambled disulfide bonds. Two additional mutant vectors encoding serine instead of half-cystine at residues 13244 and 13246 in submaxillary mucin expressed both monomers and dimers of the disulfide-rich domain but no aggregates. The final mutant vector, C13223S, expressed protein aggregates that were poorly secreted from transfected cells. A mutant vector with two codon changes, C13244A/C13246A, expressed both monomers and dimers, just like the single mutants at these half-cystines. These results suggest that three half-cystine residues (Cys13223, Cys13244, and Cys13246) may be involved in forming interchain disulfide bonds in mucin dimers. Two of these half-cystines, Cys13244 and Cys13246, are in the highly conserved sequence C13244LC13246C in the disulfide-rich domain of several other human mucins and in prepro-von Willebrand factor and norrin, a protein that in mutant forms gives rise to Norrie disease. Support for the involvement of these half-cystines in formation of disulfide-bonded dimers of these molecules is also provided by known mutations in prepro-von Willebrand factor and norrin.

Alanine↗
PubMed1997

The complete cDNA sequence and structural polymorphism of the polypeptide chain of porcine submaxillary mucin.

The complete structure of the DNA encoding the polypeptide chain of porcine submaxillary mucin has been determined. The polypeptide is composed of distinct domains. A large central domain containing tandem repeats of 81 residues each is flanked by much shorter domains with sequences similar to the tandem repeats. Four disulfide-rich domains, three at the amino terminus and one at the carboxyl terminus, complete the chain. The disulfide-rich domains have significant sequence identity to those of other mucins and prepro-von Willebrand factor. The coding region of the mucin gene is highly polymorphic, and three alleles were identified in a single animal that encoded different numbers of the 81-residue tandem repeats. A single large exon devoid of introns encodes the tandem repeat domains. The largest allele with 135 tandem repeats encoded 13,288 amino acids to give a polypeptide with Mr = 1,184,106. The other two alleles contained 99 and 125 tandem repeats, respectively. Each allele also showed different restriction fragment length polymorphisms, which is consistent with the different patterns seen in individual animals. Fragment length polymorphism was also seen within two different families of animals, indicating that the polymorphism observed occurs in a single generation.

Alleles↗
PubMed1997

Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix.

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.

Animals↗
PubMed1997

A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.

Adenosine Triphosphate↗
PubMed1997

Bactericidal and inhibitory activity of quinupristin/dalfopristin against vancomycin- and gentamicin-resistant Enterococcus faecium.

There is a need for new agents, or combinations of agents, for the treatment of infections caused by vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF) that may be resistant to all available antimicrobial agents. The early in-vitro activity of quinupristin/dalfopristin (30:70)--an injectable streptogramin--encouraged us to test this agent against VGREF. By broth dilution, the MICs of quinupristin/dalfopristin against 38 isolates of VGREF ranged from 0.06 mg/L to 2.0 mg/L (mode 0.12 mg/L). The addition of 0.5 mg/L of ciprofloxacin significantly reduced the modal MIC of quinupristin/dalfopristin to 0.015 mg/L (P = 5.75 x 10(-8)). Although the addition of 8.0 mg/L of teicoplanin or 4 mg/L of tetracycline did not significantly reduce the modal MIC, the lowest concentration of the MIC range was reduced from 0.06 to 0.015 mg/L. In broth, quinupristin/dalfopristin had slow bactericidal activity against the four strains tested over 48 h, with a 1-2 log10 cfu/mL reduction after 24 h in > 1 mg/L of quinupristin/dalfopristin for two strains and > 8 mg/L for the two other strains. A mixture of quinupristin/dalfopristin in a 70:30 ratio was more bactericidal: against one of the four strains 4-32 mg/L of the combination produced a further 0.5-1.0 log10 reduction in cfu/mL after 24 h and there was a reduction of 6.0 log10 cfu/mL after 48 h for another. By ultracentrifugation, the binding of 32 mg/L quinupristin/dalfopristin to human plasma protein was 90%, and in plasma broth, 32 mg/L of quinupristin/dalfopristin maintained bacteriostatic but not bactericidal activity. There is some useful synergy with ciprofloxacin and tetracycline, and the activity of quinupristin/dalfopristin may be enhanced against some strains by reversing the concentrations of its two components, quinupristin and dalfopristin, as that may occur in vivo.

Anti-Bacterial Agents↗