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Biomedical subjects

C Ford

Publications and source records attributed to C Ford.

At least 73 records · Page 4Linked to original sources
PubMed1994

Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation.

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.

Amino Acid Sequence↗
PubMed1994

Assessment of the intestinal retention of beta-carotene in humans.

OBJECTIVE: Assessment of the intestinal absorption of beta-carotene (BC) in humans as well as plasma clearance of BC has been difficult. We have used the total gut washout method (TGWM) to assess BC retention during transit through the intestine, as well as the effect that different diets and age have on BC retention. METHODS: HPLC was used to quantitate fecal and serum BC concentrations from young and elderly subjects who had undergone the TGWM to remove all intestinal contents prior to ingesting BC or placebo with or without a meal. Meals contained different combinations of calories and fat. RESULTS: In subjects receiving no meal, 83% of ingested BC was recovered in rectal effluent collected within 24 hours post-BC administration. The quantity of BC in feces of individuals receiving meals was 49-71%. There was no significant change in serum concentrations of other carotenoids or retinoids following consumption of BC with any of the different meals. Interestingly, both diet and age influenced the efficiency of BC absorption. An increase in dietary fat content resulted in an higher serum BC concentration in young subjects within 8 and at 24 hours post BC administration, whereas a higher caloric content resulted in a decrease in serum BC concentration in older subjects within 8 hours of BC administration. CONCLUSION: Results indicate that the TGWM provides an accurate means for assessing the intestinal retention of BC in humans.

Adolescent↗
PubMed1994

Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation.

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.

Amino Acid Sequence↗
PubMed1994

Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase.

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.

Amino Acid Sequence↗
PubMed1994

A genetic marker and family history study of the upstate New York multiple sclerosis cluster.

We report nine additional cases of new-onset multiple sclerosis (MS) among employees of an upstate New York manufacturing plant that uses zinc as a primary metal. These cases, identified during the decade 1980 to 1989, had clinical onset of the disease between 1979 and 1987. The new cases confirm the increased incidence of MS previously reported in the plant population for the 1970 to 1979 decade. The MS subjects in this occupationally based cluster do not seem different from other MS patients with regard to rates of familial MS or the frequencies of alleles for human leukocyte (HLA-DR) antigens or transferrin. The frequency distribution of alleles for transferrin (an iron- and zinc-binding protein) may differ in these and other MS subjects compared with controls.

Adult↗
PubMed1994

Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells.

Mechanisms underlying agonist- and antagonist-induced up-regulation in HEK-293 cells transfected to express D2 dopamine receptors were investigated. We have reported previously that exposure of cells to agonists and antagonists led to an increase in the density of receptors. The time course of up-regulation on exposure to (-)-sulpiride, a D2 dopamine-selective antagonist, was measured. A lag in the effect of antagonists not seen with up-regulation induced by exposure to agonists was observed. Effects of N-propylnorapomorphine were synergistic with those of cyclic AMP analogs, however, synergistic effects between (-)-sulpiride and cyclic AMP analogs were not observed. These findings suggest that separate mechanisms may be involved in agonist- and antagonist-induced up-regulation. Changes in mRNA levels did not appear to account for the increase in receptors after agonist or antagonist treatment. Results of experiments with cycloheximide, a protein-synthesis inhibitor, suggest that increased protein synthesis, and not decreased protein degradation, is responsible for up-regulation by both NPA and (-)-sulpiride. Studies monitoring receptor recovery after treatment with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, an irreversible alkylating agent, suggest that rates of receptor incorporation into membranes are increased after treatment with either an agonist or an antagonist.

Apomorphine↗
PubMed1993

Functional starch-binding domain of Aspergillus glucoamylase I in Escherichia coli.

We have fused three starch-binding domains (SBD) encoding gene fragments (residues 511-616, 495-616 and 481-616) of glucoamylase I (GAI) to the 3' end of the Escherichia coli malE gene encoding maltose-binding protein (MBP). The fusion proteins were produced in E. coli and were purified by chromatography on cross-linked amylose. Factor Xa digestion of the fusion proteins resulted in the release of functional SBD fragments which were separated from MBP on the basis of their differential binding to cross-linked amylose. The amino acid (aa) composition of the purified SBD fragments agreed with the respective amino acid compositions of GAI. The sizes of the SBD fragments were 11.9, 13.8 and 15.6 kDa, respectively.

ATP-Binding Cassette Transporters↗
PubMed1993

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis.

Fungal glucoamylases contain four conserved regions. One region from the Aspergillus niger enzyme contains three key carboxylic acid residues, the general acid catalytic group, Glu179, along with Asp176 and Glu180. Three site-directed mutations, Leu177-->His, Trp178-->Arg and Asn182-->Ala, were constructed near these acidic groups to reveal the function of other conserved residues in this region. Leu177 and Trp178 are strictly conserved among fungal glucoamylases, while an amide, predominantly Asn, always occurs at position 182. Substitutions of Leu177 or Trp178 cause significant decreases in kcat with the substrates tested. Similar increases in activation energies obtained with Leu177-->His with both alpha-(1,4)- and alpha-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178-->Arg mutation increase for an alpha-(1,6)-linked substrate, but not for alpha-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182-->Ala mutation did not change kcat or KM values, indicating that Asn182 is not crucial for activity. These results support a mechanism for glucoamylase catalytic activity consisting of a fast substrate binding step followed by a conformational change at subsite 1 to stabilize the transition state complex.

Amino Acid Sequence↗
PubMed1993

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties.

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.

Amino Acid Sequence↗
PubMed1993

Duplex ultrasonography for the detection of deep vein thrombi after total hip or knee arthroplasty.

The usefulness of real-time duplex ultrasonography (DU) as a screening test for deep vein thrombosis (DVT) in high-risk patients remains uncertain. To determine the sensitivity and specificity of DU for the detection of DVT, the authors prospectively studied 178 consecutive patients after total hip (n = 113) or total knee (n = 66) arthroplasty. The deep veins from the inguinal ligament to the ankle were examined first by continuous wave and then by pulsed Doppler signals as needed with real-time gray-scale ultrasound imaging using the criteria of vein noncompressibility to define DVT. Ascending contrast venography was performed within twelve hours after DU studies. Venograms and DU were interpreted independently. DU was attempted on 177 lower extremities (2 patients refused) but was judged adequate for interpretation for only 145 (82%). Venography could not be performed for 28 lower extremities and was technically inadequate for 8 studies. The primary analysis included 119 examinations for which adequate DU and ascending venograms were interpreted. DU was positive in 17 of 27 lower extremities with DVT (23 calf, 4 proximal) diagnosed by venography (sensitivity = .63; 95% confidence interval [CI] = .42 to .81), and DU was negative in 85 of 92 lower extremities with normal venograms (specificity = .92; 95% CI = .85 to .97). A secondary analysis of 81 prospectively collected anatomically complete DU studies demonstrated a sensitivity of .80 (95% CI = .56 to .94) and a specificity of .90 (95% CI = .80 to .96).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1992

Neonatal gross hematuria as a presenting sign of posterior urethral valves.

Neonatal gross hematuria is a rare clinical entity. Only occasionally is it associated with obstructive uropathy in the neonate. We report a case in which gross hematuria was the presenting sign in a patient with posterior urethral valves. The differential diagnosis of neonatal gross hematuria with particular attention to obstructive uropathy is reviewed.

Hematuria↗