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Biomedical subjects

K L Shaw

Publications and source records attributed to K L Shaw.

32 records · Page 2Linked to original sources
PubMed2000

Development and preliminary validation of a self-efficacy measure for use among parents of children with juvenile idiopathic arthritis.

OBJECTIVE: To develop a valid and reliable measure to assess parents' perceived ability to control, or manage, aspects of their children's juvenile idiopathic arthritis (JIA). METHODS: Construction of the Parent's Arthritis Self-Efficacy Scale (PASE) was based on existing knowledge and the results of focus groups with parents of children with JIA, children with JIA, and health professionals. Data for validation of the PASE were collected by self-administered questionnaires completed by 178 parents and 89 children with JIA. RESULTS: Analyses revealed a 2-factor structure for both mothers and fathers. These factors related to parents' self-efficacy for managing children's arthritis-related symptoms and psychosocial health. Taken together, the two factors explained 75.5% and 65.8% of the total variance (mothers' and fathers,' respectively). The PASE demonstrated high internal consistency, concurrent validity, and construct validity, particularly among mothers. CONCLUSION: Preliminary findings suggest that the PASE is worthy of further psychometric testing and may have the potential to help delineate variations in adjustment among parents of children with JIA.

Adaptation, Psychological↗
PubMed2000

Charge-charge interactions influence the denatured state ensemble and contribute to protein stability.

Several recent studies have shown that it is possible to increase protein stability by improving electrostatic interactions among charged groups on the surface of the folded protein. However, the stability increases are considerably smaller than predicted by a simple Coulomb's law calculation, and in some cases, a charge reversal on the surface leads to a decrease in stability when an increase was predicted. These results suggest that favorable charge-charge interactions are important in determining the denatured state ensemble, and that the free energy of the denatured state may be decreased more than that of the native state by reversing the charge of a side chain. We suggest that when the hydrophobic and hydrogen bonding interactions that stabilize the folded state are disrupted, the unfolded polypeptide chain rearranges to compact conformations with favorable long-range electrostatic interactions. These charge-charge interactions in the denatured state will reduce the net contribution of electrostatic interactions to protein stability and will help determine the denatured state ensemble. To support this idea, we show that the denatured state ensemble of ribonuclease Sa is considerably more compact at pH 7 where favorable charge-charge interactions are possible than at pH 3, where unfavorable electrostatic repulsion among the positive charges causes an expansion of the denatured state ensemble. Further support is provided by studies of the ionic strength dependence of the stability of charge-reversal mutants of ribonuclease Sa. These results may have important implications for the mechanism of protein folding.

Enzyme Stability↗
PubMed2000

Interspecific genetics of mate recognition: inheritance of female acoustic preference in Hawaiian crickets.

Female mating behavior plays a fundamental role in the divergent evolution of mate recognition systems that may lead to speciation. Despite this important role, the phenotypic and genetic bases of female mating behavior remain poorly understood. In this study, I examine the shape of the female acoustic preference function and estimate values for pulse rate preference in two species of Hawaiian crickets, Laupala kohalensis and L. paranigra. In addition, I examine how preference differences are inherited in hybrid crosses between these species. Females expressed unimodal preference functions and were generally more attracted to pulse rates characterizing their own species. Unimodal preference functions also characterized F1 and backcross generations, with hybrid females expressing preferences for intermediate pulse rates. Pulse rate preferences segregated in the backcross generation. Mean pulse rate preference matched mean pulse rate in both parental and hybrid generations. Based on F1 hybrids and segregation patterns in backcross females, I show that changes in both signal and receiver components of the mate recognition system are consistent with a multilocus model of change through incremental steps. The results therefore suggest that ancestors of the current species also expressed unimodal preference functions and that changes in acoustic communication signals occurred through shifts in mean pulse rates and pulse rate preferences among populations.

Acoustic Stimulation↗
PubMed2000

Characterization of the R572T point mutant of a putative cleavage site in human foamy virus Env.

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated beta-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.

Amino Acid Substitution↗
PubMed2000

The N-acetylation of arsanilic acid In vitro by mammalian enzymes.

The N-acetylation of arsanilic acid was assayed in vitro by modifying a literature method for acetylation of p-aminobenzoic acid. Conditions included final concentrations of 1.0 mM dithiothreitol, 1.0 mM EDTA, 0.45 mM acetyl coenzyme A, an acetyl coenzyme A regenerating system using bacterial phosphotransacetylase and acetyl phosphate, 5.0 mM arsanilate substrate, and 25 mM sodium/potassium phosphate buffer, pH 7.4, in a total volume of 0.5 ml. Incubation was at 37 degrees C, with 0.5- to 2-mg N-acetyltransferase enzyme protein from a preparation of guinea pig liver. The reaction was terminated by heat precipitation. The resulting supernatant was put through a 4 mm 0.45 microm polysulfone membrane syringe filter. The filtrate could then be injected directly onto the HPLC. With arsanilic acid as substrate, the product N-acetylarsanilic acid (NAA) was identified by its retention time (33 min) in the HPLC system of the laboratory. The 33-min fraction collected from the HPLC was scanned and gave the characteristic UV spectrum of NAA, with peaks at 203 and 256 nm. In addition, the product comigrated in the HPLC system with standard NAA. Under comparable assay conditions, the N-acetylation of arsanilate by the guinea pig enzyme preparation is about 24% the rate of that of the model substrate p-aminobenzoic acid. Typical activity for arsanilate acetylation was 0.5 nmol/min/mg enzyme protein. Using the same assay system and HPLC detection method, the supernatant from bacterial lysates containing recombinant human N-acetyltransferase 1 exhibited acetylation activity toward arsanilate of 720 nmol/min/mg enzyme protein.

Acetylation↗
PubMed1999

Consulting the 'experts': children's and parents' perceptions of psycho-educational interventions in the context of juvenile chronic arthritis.

There is little documented information regarding current provision of psycho-educational interventions in the context of juvenile chronic arthritis (JCA). The purpose of the present exploratory study was to gain insight and understanding of the needs and preferences of children with JCA and their parents, and to examine how these may be more adequately addressed by future psycho-educational interventions. Data were collected through focus group discussions with children, parents and health professionals, respectively. Results showed that the voices of children and their parents were as one in calling for greater availability, easier access and more comprehensive psycho-educational interventions in the context of JCA. Greater emphasis is needed upon the social and emotional aspects of chronic disease, and enhancing the self-management skills of children and their families. Moreover, content should be tailored to match broad categories of disease severity (e.g. mild or severe), developmental age and stage in the life course (e.g. child or adolescent). Group education, informal support groups, activity weekends and summer camps would be particularly welcomed by parents and children, and may serve to reduce their sense of isolation whilst providing much needed reassurance. Findings provide a useful set of signposts to guide future development of psycho-educational interventions in the field of psychosocial paediatric rheumatology.

Adult↗
PubMed1999

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.

Aspartic Acid↗
PubMed1998

Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3.

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.

Amino Acid Sequence↗
PubMed1997

A sorting motif localizes the foamy virus glycoprotein to the endoplasmic reticulum.

We recently identified an endoplasmic reticulum (ER) retrieval signal-the dilysine motif-in the glycoproteins of all five foamy viruses (FVs) for which sequences were available (P. A. Goepfert, G. Wang, and M. J. Mulligan, Cell 82:543-544, 1995). In the present study, expression of recombinant human FV (HFV) glycoprotein and analyses of oligosaccharide modifications and precursor cleavage indicated that the protein was localized to the ER. HFV glycoproteins encoding seven different dilysine motif mutations were then expressed. The results indicated that disruptions of the dilysine motif resulted in higher levels of forward transport of the HFV glycoprotein from the ER through the Golgi apparatus to the plasma membrane. We conclude that the dilysine motif is responsible for ER sorting of the FV glycoprotein. Signal-mediated ER localization has not previously been described for a retroviral glycoprotein.

Animals↗
PubMed1995

Effects of weaning, supplementation and gender on acquired immunity to Haemonchus contortus in lambs.

This study was designed to investigate whether delaying the age of weaning, or feeding a protein-rich supplement alters the rate at which lambs develop immunity to Haemonchus contortus and whether there is any interaction between nutrition, stress of weaning, and gender and the development of this immunity. Ninety-six Merino lambs were allocated to one of four treatment groups: supplemented-unweaned; supplemented-weaned; unsupplemented-unweaned; unsupplemented-weaned. There were approximately similar numbers of male and female lambs in each group. Supplemented lambs received 80 g/head/day of a protein-rich pellet from 16 to 23 weeks of age. Over the same period the lambs were drenched with 300 H. contortus larvae twice per week. Faecal worm egg counts were determined every week, and PCV and liveweight every 2 weeks for each lamb. Neither weaning nor sex had any effect on PCV (P > 0.05) but from day 50 after the start of infection, the decline in PCV was more pronounced in unsupplemented than in supplemented lambs. Faecal worm egg counts were higher (P < 0.001) in unsupplemented-weaned than in supplemented-weaned lambs and in females when compared with castrates (P < 0.03). There was a weaning x supplementation x age interaction (P < 0.03) with unsupplemented-weaned lambs developing a higher faecal egg count than supplemented-weaned lambs. There was also a significant (P < 0.02) sex x weaning x age interaction with weaned-female lambs having a higher faecal egg count than weaned-castrate lambs; egg counts increased more rapidly in unweaned-female lambs than in weaned-female lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗
PubMed1992

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1985

Changes in blood constituents accompanying exercise in polo horses.

There have been several studies of biochemical changes in horses doing intense exercise such as Thoroughbred and Standardbred racehorses and in horses performing exercise over a long period of time such as endurance horses and three-day eventing horses, but we are not aware of studies with polo horses. Blood samples were taken from 18 polo horses at rest, immediately after playing 2 chukkers of indoor polo, and after a 15 minute rest period. Each horse was studied at 2 different games. The blood samples were analyzed for lactic acid, protein, sodium, potassium, chloride, calcium, phosphorus, HCO-3, PCO2, hemoglobin, packed cell volume, and pH. Samples taken immediately after playing polo had significant increases in lactic acid, protein, sodium, hemoglobin, packed cell volume, and pH, and significant decreases in chloride, calcium, PCO2, and HCO-3. Pulse and respiration were significantly increased. After a 15 minute rest period, there was a significant decrease in potassium. The HCO-3 was lower immediately after playing, but was above the resting value after 15 minutes. It was concluded that the changes after exercise are similar in some aspects to those reported for horses performing intense exercise such as racehorses, and in some aspects to those reported for horses performing prolonged exercise such as three-day event horses and endurance horses. Horses playing indoor polo develop a high plasma lactic acid, but with alkalemia, and could be used as a model to study this condition.

Animals↗