Biomedical subjects
Publications and source records attributed to P J Reilly.
We report six cases where significant postoperative pain persisted in individuals following arthroscopic surgery augmented with the use of lasers. Subsequent magnetic resonance images showed lesions with signal changes compatible with the diagnosis of osteonecrosis in areas directly addressed with laser energy.
Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.
Isoenergy surfaces were calculated for the alpha- and beta-anomers of isomaltose and gentiobiose, based on 46,656 conformers for each disaccharide. Low-energy regions exist for each of the three staggered positions about the C-5'-C-6' bonds, and known crystal structures lie in two of these regions. As expected, the molecular partition function showed greater flexibility for these three-bond-linked disaccharides than for comparable two-bond-linked structures. A model miniature crystal of gentiobiose accounts for most of the remaining structural differences between the modeled isolated molecule and the crystal structure. Based on models of isolated molecules of isomaltose and gentiobiose, the predicted Boltzmann-weighted nmr coupling constants were satisfactory, as were predicted optical rotations for gentiobiose.
A recent article [Coutinho and Reilly (1994) Protein Engng, 7, 749-760] presented the alignment of 14 glucoamylases by hydrophobic cluster analysis. The catalytic bases of two of these glucoamylases, from Saccharomyces cerevisiae and Saccharomyces diastaticus, were not conserved, opening the possibility of a reading-frame shift error in a segment coding for amino acids near the apparent C-termini of the mature proteins. Indeed, an addition of one nucleotide restores the catalytic base, extends the sequence by 39 residues and greatly improves the amino acid alignment in this region.
Sixteen primary sequences from five sub-families of fungal, yeast and bacterial glucoamylases were related to structural information from the model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase obtained by protein crystallography. This domain is composed of thirteen alpha-helices, with five conserved regions defining the active site. Interactions between methyl alpha-maltoside and active site residues were modelled, and the importance of these residues on the catalytic action of different glucoamylases was shown by their presence in each primary sequence. The overall structure of the starch binding domain of some fungal glucoamylases was determined based on homology to the C-terminal domains of Bacillus cyclodextrin glucosyl-transferases. Crystallography indicated that this domain contains 6-8 beta-strands and homology allowed the attribution of a disulfide bridge in the glucoamylase starch binding domain. Glucoamylase residues Thr525, Asn530 and Trp560, homologous to Bacillus stearothermophilus cyclodextrin glucosyltransferase residues binding to maltose in the C-terminal domain, could be involved in raw-starch binding. The structure and length of the linker region between the catalytic and starch binding domains in fungal glucoamylases can vary substantially, a further indication of the functional independence of the two domains.
The model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase was related to 14 other glucoamylase protein sequences belonging to five subfamilies. Structural features of the different sequences were revealed by multisequence alignment following hydrophobic cluster analysis. The alignment agreed with the hydrophobic microdomains, normally conserved throughout evolution, evaluated from the 3-D model. Saccharomyces and Clostridium glucoamylases lack the alpha-helix exterior to the catalytic domain. A different catalytic base was found in the Saccharomyces glucoamylase subfamily. The starch binding domain of fungal glucoamylases has identical structural features and substrate interacting residues as the C-terminal domain of models of Bacillus circulans cyclodextrin glucosyltransferases. Three putative N-glycosylation sites were found in the same turns in glucoamylases of different subfamilies. O-Glycosylation is present at different levels in the catalytic domain and in the linker between the catalytic and starch binding domains.
Seven thermosensitive glucoamylase mutants generated by random mutagenesis and expressed in Saccharomyces cerevisiae were sequenced and their inactivation kinetics were determined. Wild-type glucoamylase expressed in S. cerevisiae was more glycosylated and more stable than the native Aspergillus niger enzyme. All mutants had lower free energies of inactivation than wild-type glucoamylase. In the Ala39-->Val, Ala302-->Val and Leu410-->Phe mutants, small hydrophobic residues were replaced by larger ones, showing that increases in size and hydrophobicity of residues included in hydrophobic clusters were destabilizing. The Gly396-->Ser and Gly407-->Asp mutants had very flexible residues replaced by more rigid ones, and this probably induced changes in the backbone conformation that destabilized the protein. The Pro128-->Ser mutation changed a rigid residue in an alpha-helix to a more flexible one, and destabilized the protein by increasing the entropy of the unfolded state. The Ala residue in the Ala442-->Thr mutation is in the highly O-glycosylated region surrounded by hydrophilic residues, where it may be a hydrophobic anchor linking the O-glycosylated arm to the catalytic core. It was replaced by a residue that potentially is O-glycosylated. In five of the seven mutations, residues that were part of hydrophobic microdomains were changed, confirming the importance of the latter in protein stability and structure.
Aspergillus awamori glucoamylase (GA) contains globular catalytic and starch-binding domains (residues 1-471 and 509-616, respectively). A heavily O-glycosylated sequence comprises two parts. The first (residues 441-471) in the crystal structure wraps around an alpha/alpha-barrel formed by residues 1-440. The second (residues 472-508) is an extended, semi-rigid linker between the two domains. To investigate the functional role of this linker, we made internal deletions to remove residues 466-512 (GA delta 1), 485-512 (GA delta 2) and 466-483 (GA delta 3). GA delta 2 and GA delta 3 were expressed in Saccharomyces cerevisiae culture supernatants at approximately 60 and 20% the wild-type level, respectively, while GA delta 1 was almost undetectable. Western blots comparing extracellular and intracellular fractions indicated that the region deleted in GA delta 3 was critical for secretion, while the region deleted in GA delta 2 contributed to the production of a stable enzyme structure. The activities of purified GA delta 2 and GA delta 3 on soluble and insoluble starch were similar to those of wild-type GA, indicating that for soluble starch their deletions did not affect the catalytic domain and for insoluble starch the linker does not coordinate the activities of the catalytic and starch-binding domains. The deletions had a significant negative effect on GA delta 2 and GA delta 3 thermostabilities.
Nine single amino acid mutations in the active site of Aspergillus awamori glucoamylase were made by cassette mutagenesis to alter the pH dependence of the enzyme and to determine possible functions of the mutated residues. The Glu179-->Asp mutation expressed in yeast led to a very large decrease in kcat but to no change in Km, verifying this residue's catalytic function. Asp176-->Glu and Glu180-->Asp mutations affected Km more than kcat, implying that Asp176 and Glu180 are involved in substrate binding or structural integrity. The Leu177-->Asp mutation decreased kcat only moderately, probably by changing the position of the general acid catalytic group, and did not affect Km. The Trp178-->Asp mutation greatly decreased kcat while increasing Km, showing the importance of Trp178 in the active site. Val181-->Asp and Asn182-->Asp mutations changed kinetic values little, suggesting that Val181 and Asn182 are of minor catalytic and structural importance. Finally, insertions of Asp or Gly between residues 176 and 177 resulted in almost complete loss of activity, probably caused by destruction of the active site structure. No large changes in pH dependence occurred in those mutations where kinetic values could be determined, in spite of the increase in most cases of the total negative charge. Increases in activation energy of maltoheptaose hydrolysis in most of the mutant glucoamylases suggested cleavage of individual hydrogen bonds in enzyme-substrate complexes.
Relaxed-residue energy maps based on the MM3 force-field were computed for relative orientations of the pyranosyl rings of sophorose, laminarabiose, and cellobiose, respectively the (1----2)-beta-; (1----3)-beta-; and (1----4)-beta-linked D-glucosyl disaccharides. Sixteen starting conformations of the rotatable exocyclic side-groups were considered for each molecule. All of the energy surfaces have two intersecting low-energy troughs and illustrate the importance of exo-anomeric effects in determining disaccharide conformation. Local minima were found by relaxed minimization without restriction. The energy surfaces of these disaccharides are very similar to the energy surfaces of their corresponding 6-methyltetrahydropyran analogues. There is good agreement between disaccharide structures having minimal MM3 energy and those found by crystallography.
Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1----2)-alpha, (1----3)-alpha, and (1----4)-alpha-linked D-glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20 degree grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.
The occurrence of Salmonella and Vibrio cholerae in brackishwater ponds was monitored over a 2-year period in one of the major prawn exporting countries in Southeast Asia. The principal production areas were identified and regular samples taken for Salmonella and V. cholerae analysis. Results demonstrated that brackishwater ponds and cultured prawns were inherently contaminated with both bacterial pathogens. Salmonella spp. were present in 16.0% of prawns and 22.1% of mud/water samples from ponds; and V. cholerae present in 1.5% of prawns and 3.1% of mud/water samples. Culturing by intensive methods tended to favour contamination by these pathogens, which is most likely due to the accumulation of waste and increase in the volume of sediments in ponds. Typical environmental factors such as water temperature, pH, and salinity were all favourable for growth of microorganisms. The incidence of the pathogens increased during the wet season and was marginally higher when ponds were located close to urban areas. S. weltevreden was identified as the principal serotype found in ponds, and to a lesser extent S. anatum (11%) S. wandsworth (8%) and S. potsdam (8%). The V. cholerae belonged to the non-O1 serogroup.
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We investigated whether a volatile anesthetic (1.5% isoflurane or 1.0% halothane) or an added anaerobic energy source (10 mM glucose or fructose) could act directly on liver cells to protect energy status during 20-30 min of anoxia. We used hepatocytes freshly isolated from fed rats or rats that had fasted, suspended them in Krebs' buffer, and incubated them in sealed flasks under O2/CO2 or N2/CO2 (95%:5%). The adenosine triphosphate (ATP) to adenosine diphosphate (ADP) ratio (ATP/ADP) measured cellular energy balance--the balance between overall ATP supply and demand. Lactate levels measured the extent to which ATP was supplied by the nonmitochondrial pathway, (anaerobic) glycolysis. Maximum values of energy balance were seen in cells from fed rats incubated in the presence of glucose and O2. When glucose was replaced by fructose, ATP/ADP decreased and lactate increased. During anoxia (O2 replaced by N2), increases in lactate were also seen with glucose; and ATP/ADP decreased to similarly low values with both substrates. In cells from fasted rats, ATP/ADP decreased significantly below the value for cells from fed rats only in the presence of glucose and O2. Compared with cells from fed rats, cells from fasted rats showed decreased lactate in the face of decreased ATP/ADP, suggesting that glycolysis was impaired. Isoflurane partially prevented anoxia-induced decreases in ATP/ADP. This protective effect on energy balance occurred equally with glucose and fructose, but was not seen in cells from fasted rats or with halothane. Thus, 1 MAC isoflurane and some factor(s) related to the fed state combined to protect partially the energy balance in anoxic liver cells through action(s) at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)
Excessive Ca2+ influx through NMDA receptor-coupled channels has been linked to neuronal cell death. Using an in vitro model of transient brain ischemia, we investigated possible protective effects of NMDA receptor antagonists ketamine or MK-801 and of calmidazolium, an inhibitor of intracellular Ca2(+)-activated proteins. Brain ischemia/recovery was simulated in isolated hippocampal slices and injury monitored by measurement of ATP levels. Omission of both glucose and oxygen (but not oxygen alone) for 20 min led to persistent ATP deficits after 4 h recovery. Addition of ketamine or MK-801 at 1 microM permitted ATP to recover within 1 h, as did addition of calmidazolium at 10 microM. Our findings are consistent with other reports that NMDA receptor antagonists can protect neuronal tissue from ischemic damage. The role of inappropriately activated Ca2(+)-mediated signaling processes in the mechanism(s) of such injury is suggested by the protection also seen with calmidazolium, an inhibitor of calmodulin and other structurally related proteins such as calpain(s) and protein kinase C. The inhibition of intracellular Ca2+ target proteins may be an alternative for protection of the brain against injury due to insults that activate NMDA receptors.
Eight monosaccharides (L-arabinoside, L-fucose, D-galactose, D-glucose, D-lyxose, D-mannose, L-rhamnose, and D-xylose), eight disaccharides (cellobiose, gentiobiose, isomaltose, lactose, maltose, nigerose, sophorose, and xylobiose), and three trisaccharides (isomaltotriose, maltotriose, and xylotriose) were derivatized with N-methylbis-(trifluoroacetamide) in pyridine solution to form trifluoroacetylated derivatives. These were analyzed by 1H-n.m.r. spectroscopy to determine the characteristics of the spectra and distributions of the reaction products. Peaks corresponding to reducing-residue anomeric protons were located significantly downfield of all others, and were in general 0.4 p.p.m. or more downfield of equivalent signals from the same carbohydrates when they were free or derivatized with other groups. Neither the location of anomeric proton peaks relative to each other nor the degree of spin-spin coupling between H-1 and H-2 varied greatly with type of derivatization. Spin-spin coupling, however, decreased for some beta-pyranose forms of xylobiose and the three trisaccharides. In all examples except some where H-2 was oriented equatorially to a pyranose ring, the proportion of the alpha-pyranose was either enhanced or not changed in concentration by trifluoroacetylation.
Dextromoramide (Palfium) was given by intravenous injection to a Thoroughbred horse at a dosage of 20 mg and urine was collected 2, 4, 6 and 8 h after drug administration. Enzymatic hydrolysis of the urine followed by solvent extraction gave a residue which was back-extracted into 0.1 M sulphuric acid. After basification to pH 9 and solvent extraction, the resulting residue was submitted to gas chromatographic-mass spectrometric analysis. Both electron-impact and ammonia chemical-ionization mass spectra were recorded and, based on the observed fragmentation patterns, the principal metabolites in horse urine were shown to be 2,2-diphenyl-3-methyl-4-morpholinobutyramide (compound 2) and the product of hydroxylation of one phenyl ring in dextromoramide (compound 3), respectively. The electron-impact mass spectra of compounds 2 and 3, and of their derivatisation products from oncolumn methylation in the gas chromatograph, are reported.
Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylase appeared by differential labeling to be in the active site. To test their functions, they were replaced by mutagenesis with Asn, Gln and Gln respectively, and kinetic parameters and pH dependencies of all enzyme forms were determined. Glu179----Gln glucoamylase was not active on maltose or isomaltose, while the kcat for maltoheptaose hydrolysis decreased almost 2000-fold and the KM was essentially unchanged from wild-type glucoamylase. The The Glu180----Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Difference in substrate activation energies between Glu180----Gln and wild-type glucoamylases indicate that Glu180 binds D-glucosyl residues in subsite 2. The Asp176----Asn substitution gave moderate increases and decreases in KM and kcat respectively, and therefore similar increases in activation energies for the three substrates. This and the differences in subsite binding energies between Asp176----Asn and wild-type glucoamylases suggest that Asp176 is near subsite 1, where it stabilizes the transition state and interacts with Trp120 at subsite 4. Glu179 and Asp176 are thus proposed as the general catalytic acid and base of pKa 5.9 and 2.7 respectively. The charged Glu180 contributes to the high pKa value of Glu179.