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Biomedical subjects

L Wells

Publications and source records attributed to L Wells.

At least 37 records · Page 2Linked to original sources
PubMed1996

Functional requirements of the active site position 185 in the human enzyme galactose-1-phosphate uridylyltransferase.

The active site of galactose-1-phosphate uridylyltransferase (GALT) includes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans. The crystal structure of the E. coli enzyme suggests that this proline is important in positioning the active site histidine (His-166) near the substrate. To examine the role of this proline in the homologous human sequence, we have performed saturating mutagenesis at Pro-185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system. Activity analyses in crude lysates indicated that only proline at position 185 produced wild-type levels of activity, although five other amino acids, Ala, Gly, Ser, Gln, and Glu, all produced partially active enzymes. Western blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity. All five active mutant proteins were purified and characterized with regard to specific activity, apparent Km for both substrates, and temperature-dependence of activity. Finally, modeling of these mutations onto the conserved E. coli active site structure was performed. Together, these results provide functional evidence demonstrating the critical role of Pro-185 in facilitating the transferase reaction.

Binding Sites↗
PubMed1996

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.

Animals↗
PubMed1996

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Alleles↗
PubMed1996

Relation of systemic and local muscle exercise capacity to skeletal muscle characteristics in men with congestive heart failure.

OBJECTIVES: The present study was undertaken to further characterize changes in skeletal muscle morphology and histochemistry in congestive heart failure and to determine the relation of these changes to abnormalities of systemic and local muscle exercise capacity. BACKGROUND: Abnormalities of skeletal muscle appear to play a role in the limitation of exercise capacity in congestive heart failure, but information on the changes in muscle morphology and biochemistry and their relation to alterations in muscle function is limited. METHODS: Eighteen men with predominantly mild to moderate congestive heart failure (mean +/- SEM New York Heart Association functional class 2.6 +/- 0.2, ejection fraction 24 +/- 2%) and eight age- and gender-matched sedentary control subjects underwent measurements of peak systemic oxygen consumption (VO2) during cycle ergometry, resistance to fatigue of the quadriceps femoris muscle group and biopsy of the vastus lateralis muscle. RESULTS: Peak VO2 and resistance to fatigue were lower in the patients with heart failure than in control subjects (15.7 +/- 1.2 vs. 25.1 +/- 1.5 ml/min-kg and 63 +/- 2% vs. 85 +/- 3%, respectively, both p < 0.001). Patients had a lower proportion of slow twitch, type I fibers than did control subjects (36 +/- 3% vs. 46 +/- 5%, p = 0.048) and a higher proportion of fast twitch, type IIab fibers (18 +/- 3% vs. 7 +/- 2%, p = 0.004). Fiber cross-sectional area was smaller, and single-fiber succinate dehydrogenase activity, a mitochondrial oxidative marker, was lower in patients (both p < or = 0.034). Likewise, the ratio of average fast twitch to slow twitch fiber cross-sectional area was lower in patients (0.780 +/- 0.06 vs. 1.05 +/- 0.08, p = 0.019). Peak VO2 was strongly related to integrated succinate dehydrogenase activity in patients (r = 0.896, p = 0.001). Peak VO2, resistance to fatigue and strength also correlated significantly with several measures of fiber size, especially of fast twitch fibers, in patients. None of the skeletal muscle characteristics examined correlated with exercise capacity in control subjects. CONCLUSIONS: These results indicate that congestive heart failure is associated with changes in the characteristics of skeletal muscle and local as well as systemic exercise performance. There are fewer slow twitch fibers, smaller fast twitch fibers and lower succinate dehydrogenase activity. The latter finding suggests that mitochondrial content of muscle is reduced in heart failure and that impaired aerobic-oxidative capacity may play a role in the limitation of systemic exercise capacity.

Aged↗
PubMed1996

Common lower extremity problems in children.

This article discusses the diagnosis and management of common lower extremity problems in children, including flatfeet, torsional and angular problems, developmental dislocation of the hip, and slipped capital femoral epiphysis. These problems represent some of the more common lower extremity conditions found in children from infancy to adolescence.

Child↗
PubMed1996

A memorial service for families of children who died from cancer and blood disorders.

The grief of staff who work with dying children and the grief of family members after the death of a child has been widely documented. Interventions to facilitate grieving have been extensively developed for parents but less so for siblings and staff. This article describes one approach, a memorial service, for families and staff that has wide applicability for providing support after a death. The memorial service provides families and staff with a healing ritual of remembrance, a source of closure after the death, and a recognition of the relationships established between families and staff. The service particularly legitimizes the staff's grief experience. The organization, implementation, and evaluation of such a program is discussed.

Adult↗
PubMed1996

The yeast, Saccharomyces cerevisiae, as a model system for the study of human genetic disease.

Many human genes associated with disease have close homologs in yeast. Based on this homology, many human proteins have been studied using yeast expression systems. This paper will review research done in our laboratory using a yeast expression system to study the human protein galactose-1-phosphate uridylyltransferase, associated with galactosemia, as well as highlighting some of the advantages of this model system.

Galactosemias↗
PubMed1995

Relation of the nine-minute self-powered treadmill test to maximal exercise capacity and skeletal muscle function in patients with congestive heart failure.

The 9-minute self-powered treadmill test has been employed to evaluate submaximal exercise capacity in heart failure patients, but its relation to maximal exercise capacity and to indexes of skeletal muscle function has not been well defined. Two protocols were utilized. The first evaluated the relation of the peak oxygen uptake (VO2) achieved on the self-powered treadmill to that during a symptom-limited treadmill protocol, and examined the reproducibility of this test. Thirteen patients (aged 62 +/- 2 years, in New York Heart Association class I to III [2.3 +/- 0.1], ejection fraction 23 +/- 2% [means +/- SEM]) and 10 age-matched sedentary controls were studied. The second protocol, which involved 18 patients (aged 65 +/- 2 years, in New York Heart Association class I to IV [2.4 +/- 0.1], ejection fraction 23 +/- 2%) and 10 age-matched controls evaluated the relation of performance on the self-powered treadmill to maximal systemic exercise capacity on a cycle ergometer and to indexes of skeletal muscle function. In the first protocol, the test was found to be highly reproducible. The proportion of self-powered treadmill to maximal treadmill peak VO2 did not differ significantly between patients and controls (95 +/- 5% vs 87 +/- 6%). In the second protocol, patients achieved a lower peak VO2 (15.6 +/- 1.1 vs 25.6 +/- 0.9 ml/kg/min, p < 0.001), walked a shorter distance on the self-powered treadmill (367 +/- 32 vs 667 +/- 28 m, p < 0.001), and exhibited less knee extensor work capacity (1,075 +/- 116 vs 1,390 +/- 110 ft-lbs, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system.

Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT.

Alleles↗
PubMed1995

Exercise intolerance in chronic heart failure is not associated with impaired recovery of muscle function or submaximal exercise performance.

OBJECTIVES: This study investigated whether recovery of skeletal muscle function is impaired in patients with heart failure and whether impaired recovery is associated with abnormal submaximal systemic exercise tolerance during repeated testing. BACKGROUND: Patients with heart failure experience fatigue during daily activities. Because abnormalities of skeletal muscle play a role in their exercise intolerance, these symptoms may reflect a delay in muscle recovery and a resulting limitation in submaximal exercise tolerance. METHODS: Two protocols were used. In protocol 1, knee extensor strength and endurance, and their recovery after fatiguing exercise, were evaluated in 11 patients (mean [+/- SEM] age 62 +/- 5 years, New York Heart Association functional class 2.3 +/- 0.2, ejection fraction 24 +/- 5%) and in 10 age-matched sedentary control subjects. Protocol 2 examined the recovery of knee extensor endurance and submaximal exercise tolerance, as quantified on a self-powered treadmill, over 24 h in 18 patients (mean age 65 +/- 3 years, functional class 2.4 +/- 0.2, ejection fraction 23 +/- 3%) and in 10 control subjects. RESULTS: Peak oxygen consumption was reduced in both heart failure groups (15.4 +/- 1.4 and 15.6 +/- 1.0 ml/kg per min) compared with that in the respective control groups (23.1 +/- 2.9 and 25.6 +/- 1.0 ml/kg per min, both p < 0.05), as was muscle endurance but not muscle strength. In protocol 1, knee extensor endurance recovered more slowly in the patients than in control subjects (to 62 +/- 4% and 87 +/- 7% of the baseline value after 5 min, respectively, p < 0.05). In protocol 2, submaximal exercise tolerance was lower in the patients with heart failure than in control subjects (1,075 +/- 116 vs. 1,390 +/- 110 m), but knee extensor endurance and walking distance recovered fully by 10 and 30 min, respectively. CONCLUSIONS: Although these findings confirm earlier studies that demonstrated impaired muscle endurance in patients with heart failure, the results provide no evidence that recovery of either muscle function or submaximal exercise tolerance is delayed beyond the initial 5 to 10 min after exercise.

Aged↗
PubMed1995

Insulin-like growth factor 1 and functional status in healthy older men.

OBJECTIVE: To determine if insulin-like growth factor 1 (IGF-1) is associated with strength and functional ability in healthy older men. DESIGN: Cross-sectional study. SETTING: San Francisco Department of Veterans Affairs Medical Center. PARTICIPANTS: One hundred four ambulatory community-dwelling men. MEASUREMENTS: Serum IGF-1 levels were obtained. Measured variables included strength of the knee flexors and extensors, handgrip, score on the Physical Performance Test, body composition, and three tests of cognitive function. RESULTS: The subjects' mean age was 75.5 +/- 4.9 (SD) years (range 70-94 years), and their mean IGF-1 level was 134.7 +/- 43.6 ng/mL. The univariate association of age with the variables was much stronger than the univariate association of IGF-1 with the same variables. In multivariable models, age, but not IGF-1, was associated with the variables. CONCLUSION: In this study of healthy older men, age is the most important variable in predicting functional decline. There was no association of IGF-1 levels to functional status independent of age.

Activities of Daily Living↗
PubMed1995

Corticosteroid-binding globulin in preterm infants in an intensive care unit.

Blood concentration of corticosteroid-binding globulin (CBG) was measured in 85 preterm (24-36 weeks of gestational age) infants over the first 6 weeks of postnatal life. CBG concentrations directly correlated to gestational age and postnatal age in the first week of life. There was a significant increase in CBG values over the first 6 weeks such that mean values at weeks 4 through 6 were significantly greater than values at weeks 1 and 2. Postnatal CBG values were significantly lower in infants who had pulmonary disease during week 1 (p = 0.001). In summary, CBG values were directly correlated to gestational and postnatal age, and negatively correlated to acute illness. We speculate that the lower CBG values with lung disease may reflect an ongoing inflammatory response.

Aging↗
PubMed1993

A clinical therapeutic trial of cyclosporine in myasthenia gravis.

We randomly assigned 39 patients with steroid-dependent generalized myasthenia gravis to treatment with cyclosporine (5 mg/kg per body weight in divided doses) or placebo. Duration of treatment was 6 months. Patients were evaluated monthly. Primary measures of efficacy were quantified strength testing, antihuman acetylcholine receptor antibody titer, and dosage of corticosteroid medication. At the end of the study, patients in the cyclosporine group had significantly greater improvement in strength (p = 0.004) and a reduction in antireceptor antibody titer (p = 0.01). Percentage reduction of steroid medication was greater in the cyclosporine group, although the difference was not statistically significant (p = 0.12). There were no treatment failures, and there was one drug failure in the cyclosporine group. In the placebo group, there were three treatment failures. No significant nephrotoxicity was noted at this dosage during the first 6 months. During the subsequent 18 months of open-label therapy, continued reduction in steroid dosage occurred. Cumulative side effects, however, caused 35% of patients to discontinue the medication; 10% did so secondary to slowly progressive nephrotoxicity.

Adult↗
PubMed1993

Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity.

Enzymatically active and inactive (diisopropylfluorophosphate-treated) cathepsin G exerted antibacterial action in vitro against Staphylococcus aureus, whereas only enzymatically active cathepsin G displayed bactericidal action against Pseudomonas aeruginosa. In order to further test the requirement for protease activity for the antipseudomonal action of cathepsin G, synthetic peptides spanning the full-length mature protein were prepared and examined for antibacterial action. Surprisingly, three structurally distinct peptides that correspond to residues 61 to 80, 117 to 136, and 198 to 223 within the full-length protein were found to exert potent antipseudomonal action (> 4.5 logs of killing at 500 micrograms/ml) against P. aeruginosa ATCC 27853 and four mucoid clinical isolates. Only the peptide (CG117-136) corresponding to residues 117 to 136 (117-RPGTLCTVAGWGRVSMRRGT-136) within cathepsin G exerted antibacterial action against the gram-positive pathogen S. aureus. The antipseudomonal action of CG117-136 was rapid and could be inhibited either by increasing concentrations of NaCl or by 0.5 mM MgCl2 plus 0.5 mM CaCl2, and these conditions appeared to reduce binding of the peptide to whole bacteria. Variants of peptide CG117-136 lacking either a hydrophobic N-terminal domain or a positively charged C-terminal domain were found to have significantly less antipseudomonal action than CG117-136. The antibacterial capacity of the all-D-enantiomeric form of peptide CG117-136 was found to be identical to that of the all-L-peptide, suggesting that the mechanism of killing does not require the recognition of a target site possessing a chiral center.

Amino Acid Sequence↗
PubMed1993

Skeletal muscle size: relationship to muscle function in heart failure.

Abnormalities of skeletal muscle function and metabolism are common in patients with congestive heart failure (CHF) and appear to contribute to systemic exercise limitation. Although the mechanism for these differences is unclear, one possibility is skeletal muscle atrophy. In 21 CHF patients and 12 sex- and age-matched sedentary control subjects, we quantified muscle size as maximal cross-sectional area (MCSA) of thigh muscles measured by magnetic resonance imaging and determined the relationship between muscle size and muscle function. Muscle strength was measured as maximum force developed during isometric contractions, and muscle endurance was quantified as the decline in force during 15 consecutive isokinetic knee extensions (measured as ratio of mean peak torque of last 3 and first 3 extensions). MCSAs of thigh muscles (141 +/- 28 vs. 167 +/- 47 cm2, P < 0.05) and knee extensors (62 +/- 13 vs. 75 +/- 13 cm2; P < 0.05) were both significantly smaller in patients than in control subjects. These differences persisted after normalization for body size. Isometric strength was less, but not significantly so, in patients (126 +/- 39 vs. 135 +/- 43 Nm; P = NS), but muscle endurance was markedly impaired (endurance ratio 0.67 +/- 0.14 vs. 0.83 +/- 0.11; P < 0.05). A strong correlation was found between isometric strength (r = 0.76) and MCSA of knee extensors, but only a weak correlation between dynamic endurance and MCSA was seen. We conclude that muscle size is smaller in CHF patients but that maximal force generated per area of muscle is not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

High-risk indicators for family involvement in social work in health care: a review of the literature.

Shifts in health concerns, fiscal restraints, technological advances, and demands for accountability have created severe tensions within health care settings. New demands point to the need for a redefinition of services. High-risk screening appears to be a clear method of delineating service need. A review of the empirical literature highlights individual, family, and illness variables that alone and together may improve identification of patients and families in need of social work services. The development of screening mechanisms may be a useful vehicle for improved psychosocial care and for the planning of social work services.

Canada↗