Burst inflation test for measuring biomechanical properties of rat abdominal walls.

PURPOSE: Evaluation of potential grafts to improve upon current strategies for abdominal wall (AW) repair in small animal models typically involves mechanical testing using methods that currently are inadequate to assess physiologically relevant parameters. This study introduces burst inflation testing as a more relevant assessment of the mechanical integrity of the AW compared to traditional tensile testing. METHODS: AWs were excised from 14 healthy adult Fischer 344 rats and tested using either a custom burst inflation device or an Instron tensile testing system. Modulus outcomes from both testing methods were compared. RESULTS: Mechanical analyses of native AW using burst and tensile testing methods resulted in similar average tissue moduli, but with the burst test, there was significantly less variability among specimens. CONCLUSIONS: The burst test had greater repeatability compared to tensile testing and has the ability to test repaired AWs without compromising the integrity of the repair site, making it a useful tool for assessing graft repairs.

Controlled outcome evaluation of the First Step Program: a daily physical activity intervention for individuals with type II diabetes.

OBJECTIVE: To conduct a randomised trial of a physical activity (PA) intervention, The First Step Program (FSP) for adults with type II diabetes. DESIGN: A 16-week intervention study and 24-week follow-up assessment. PARTICIPANTS: A total of 47 overweight/obese, sedentary individuals (age=52.7 +/- 5.2 y; BMI=33.3 +/- 5.6 kg/m2) recruited through a diabetes education centre. PRIMARY OUTCOME: daily PA assessed by pedometer (steps/day). SECONDARY OUTCOMES: anthropometric measures (weight, BMI, waist girth, hip girth); indicators of cardiovascular health (resting heart rate and blood pressure); glycemic control (fasting glucose, insulin, HbA1c, glucose concentration 120 min postglucose load); plasma lipid status (total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides). RESULTS: Relative to the CONTROL group, FSP participants increased their PA >3000 steps/day (approximately 30 min/day) during the intervention (P<0.0001). Waist and hip girth decreased (approximately 2-3 cm), but did not differ significantly between groups. Significant changes did not emerge for any of the other variables. CONCLUSIONS: The FSP is a practical intervention that elicits an immediate and profound change in walking behaviour. Such change is an important 'first step' towards increasing the volume and/or intensity of PA necessary to improve long-term health outcomes in this largely sedentary and overweight or obese population. Relapse by 24 weeks indicates that other strategies such as booster sessions are needed to maintain lifestyle change. Further research must determine realistic and responsive health outcomes for this population that are achievable through practical, real-world programming.

Alteration in goblet cell numbers and mucin gene expression in a mouse model of allergic conjunctivitis.

PURPOSE: To determine whether the number of filled conjunctival goblet cells and mucin gene expression are altered in a mouse model of allergic conjunctivitis. METHODS: A/J mice were sensitized intraperitoneally with cat dander or the peptide P3-1 from the protein Fel d1. Two weeks later, the mice were challenged for 7 consecutive days with eye drops containing the allergens. Conjunctival tissue was harvested at 0, 6, 24, or 48 hours after final antigen challenge. Control samples were naïve animals and mice sensitized with cat dander and challenged with OVA-peptide or PBS. The mean number of filled goblet cells per square millimeter in three forniceal fields for each group was determined in wholemounts of conjunctiva prepared using rhodamine-phalloidin labeling followed by confocal microscopy. RNA was isolated from conjunctiva of the contralateral eye and taken for relative quantitation of mRNA of the goblet cell mucin Muc5AC and the epithelial membrane-spanning mucin Muc4, by real-time RT-PCR. RESULTS: The number of filled goblet cells was significantly decreased with both cat dander and P3-1, after final ocular challenge (P < 0.001). The most significant decrease over naïve mice was seen at 6 hours after final challenge with both allergens. The number of filled goblet cells was still decreased but was returning toward naïve levels at 24 hours (P < 0.05), and at 48 hours no significant difference was seen compared with naïve, PBS-treated, and OVA-peptide-treated control samples. For both cat dander and P3-1, Muc5AC and Muc4 mRNA was found to be decreased at the time of final ocular challenge. The level of Muc5AC mRNA from goblet cells rebounded from the decrease to show an increase over control by 24 hours after final challenge, and by 48 hours, the mRNA level had returned to naïve control range. In contrast, significant increases in Muc5AC mRNA were evident after final control challenge with PBS or OVA-peptide, indicating a potential irritant effect of drop application. The Muc4 mRNA level was significantly reduced at all time points except 24 hours after the last challenge. By comparison with allergen-challenged eyes, no change in Muc4 message levels was noted at any time point in OVA-peptide- or PBS-treated control eyes. CONCLUSIONS: These findings demonstrate that, in the conjunctiva of mice, repetitive application of allergens induces a reduction in the number of filled goblet cells and a decrease in Muc5AC and Muc4 mRNAs. After a period of 24 to 48 hours, the goblet cell number return to naïve levels, and goblet cell mucin mRNA levels return to above or within normal range, indicating a rapid recovery in the mucus secretion system.

Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.

Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation.

Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.

Challenges and opportunities for measuring physical activity in sedentary adults.

Increasing the physical activity of typically sedentary adult populations is at the forefront of the public health agenda. This review addresses the challenges in defining and measuring physical activity in this target group, for a number of purposes, namely, scientific or academic inquiry, surveillance, clinical application and programme evaluation. First, we clarify the conceptual distinctions between the terms sedentarism, physical inactivity, physical activity and energy expenditure. Next, we review and compare the utility of different approaches for quantifying and expressing physical activity in these populations. Physical activity in typically sedentary populations is most likely a simple pattern of behaviour that has been largely obscured by existing measures and its expression as energy expenditure. Existing self-report methods are practical, but suffer from floor effects and recall bias. Walking, the most important activity to assess in this target group, is very difficult to measure through self-report methods. Motion sensors are more appropriate for quantifying physical activity behaviours in typically sedentary populations. Of the 2 types of motion sensors - the accelerometer and the pedometers--the latter is more appealing because it is both an affordable and a 'good enough' measure of physical activity, specifically ambulatory activity. Although a common measurement approach would greatly facilitate our understanding of physical activity behaviour patterns, the selection of an approach ultimately depends on the purpose of the study and to a great extent, its budget. Researchers, clinicians and practitioners interested in accurately capturing the lower end of the continuum of physical activity (that is characteristic of sedentary populations) must thoughtfully consider the relative advantages and disadvantages of the available approaches.

Methodological considerations for researchers and practitioners using pedometers to measure physical (ambulatory) activity.

Researchers and practitioners require guidelines for using electronic pedometers to objectively quantify physical activity (specifically ambulatory activity) for research and surveillance as well as clinical and program applications. Methodological considerations include choice of metric and length of monitoring frame as well as different data recording and collection procedures. A systematic review of 32 empirical studies suggests we can expect 12,000-16,000 steps/day for 8-10-year-old children (lower for girls than boys); 7,000-13,000 steps/day for relatively healthy, younger adults (lower for women than men); 6,000-8,500 steps/day for healthy older adults; and 3,500-5,500 steps/day for individuals living with disabilities and chronic illnesses. These preliminary recommendations should be modified and refined, as evidence and experience using pedometers accumulates.

Molecular structure of three mutations at the maize sugary1 locus and their allele-specific phenotypic effects.

Starch production in all plants examined is altered by mutations of isoamylase-type starch-debranching enzymes (DBE), although how these proteins affect glucan polymer assembly is not understood. Various allelic mutations in the maize (Zea mays) gene sugary1 (su1), which codes for an isoamylase-type DBE, condition distinct kernel phenotypes. This study characterized the recessive mutations su1-Ref, su1-R4582::Mu1, and su1-st, regarding their molecular basis, chemical phenotypes, and effects on starch metabolizing enzymes. The su1-Ref allele results in two specific amino acid substitutions without affecting the Su1 mRNA level. The su1-R4582::Mu1 mutation is a null allele that abolishes transcript accumulation. The su1-st mutation results from insertion of a novel transposon-like sequence, designated Toad, which causes alternative pre-mRNA splicing. Three su1-st mutant transcripts are produced, one that is nonfunctional and two that code for modified SU1 polypeptides. The su1-st mutation is dominant to the null allele su1-R4582::Mu1, but recessive to su1-Ref, suggestive of complex effects involving quaternary structure of the SU1 enzyme. All three su1- alleles severely reduce or eliminate isoamylase-type DBE activity, although su1-st kernels accumulate less phytoglycogen and Suc than su1-Ref or su1-R4582::Mu1 mutants. The chain length distribution of residual amylopectin is significantly altered by su1-Ref and su1-R4582::Mu1, whereas su1-st has modest effects. These results, together with su1 allele-specific effects on other starch- metabolizing enzymes detected in zymograms, suggest that total DBE catalytic activity is the not the sole determinant of Su1 function and that specific interactions between SU1 and other components of the starch biosynthetic system are required.

Development of a theory-based daily activity intervention for individuals with type 2 diabetes.

PURPOSE: This article describes a theory-driven approach to developing a physical activity intervention for sedentary individuals with type 2 diabetes. METHODS: Development of the intervention was based on 6 essential elements of program theory: problem definition, critical inputs, mediating processes, expected outcomes, extraneous factors, and implementation issues. Each element was formulated based on available literature and in collaboration with both intended service deliverers (diabetes educators) and recipients (sedentary persons with type 2 diabetes). RESULTS: Diabetes education requires a simple physical activity intervention template that is feasible, acceptable, and effective in a variety of settings. Successful programs are individualized, specific, flexible, and based on walking. Pedometers have potential as self-monitoring and feedback tools. The primary expected outcome is an increase in physical activity, specifically walking. Behavior modification and social support are critical to adoption and adherence. CONCLUSIONS: Theory-driven interventions specify what works for whom and under what conditions of delivery. The underlying theory guides the evaluation, refinement, and clinical replication of an intervention. Recruitment, delivery, and follow-up are real-world implementation issues.

Relationship between choice and quality of life among residents in long-term-care facilities.

OBJECTIVE: This study tested the association between perceptions of personal control and quality of life among older persons. METHOD: Two self-report instruments. The Quality of Life Rating (QOLR) and the Duncan Choice Index (DCI), were administered to 21 residents in a long-term-care facility. The DCI was developed for this study to measure the amount of choice available in 29 self-care and leisure activities. RESULTS: A significant positive correlation (r = .54; p = .01) between the amount of choice residents perceive they have and their quality of life was found. The DCI was shown to be reliable with preliminary evidence of construct validity. CONCLUSION: Enhancing personal control in everyday life may be associated with improved quality of life. Occupational therapy strategies to empower residents through increasing choice and control include increasing community in the facility emphasizing personal responsibility, and enabling choices in everyday tasks.

Identification and characterization of a critical CP2-binding element in the human interleukin-4 promoter.

Expression of cytokine genes in T cells is thought to result from a complex network of antigen- and mitogen-activated transcriptional regulators. CP2, a factor homologous to Drosophila Elf-1 and previously found to be a critical regulator of several viral and cellular genes in response to developmental signals, is rapidly activated in T helper (Th) cells in response to mitogenic stimulation. Here we show that overexpression of CP2 enhances interleukin (IL)-4 promoter-driven chloramphenicol acetyltransferase expression, while repressing IL-2 promoter activity, in transiently transfected Jurkat cells. A CP2-protected element, partially overlapping the nuclear factor of activated T cell-binding P2 sequence, was required for IL-4 promoter activation in CP2-overexpressing Jurkat cells. This CP2-response element is the site of a cooperative interaction between CP2 and an inducible heteromeric co-factor(s). Mutation of conserved nucleotide contacts within the CP2-response element prevented CP2 binding and significantly reduced constitutive and induced IL-4 promoter activity. Expression of a CP2 mutant lacking the Elf-1-homology region of the DNA-binding domain inhibited IL-4 promoter activity in a dominant negative fashion in transiently transfected Jurkat cells. Moreover, overexpressed CP2 markedly enhanced, while its dominant negative mutant consistently suppressed, expression of the endogenous IL-4 gene in the murine Th2 cell line D10. Taken together, these findings point to CP2 as a critical IL-4 transactivator in Th cells.

Purification and characterization of soluble starch synthases from maize endosperm.

This study identified and characterized the soluble starch synthase of maize endosperm that was initially revealed as the SSII activity peak in anion exchange chromatography (J. L. Ozbun et al. (1971) Plant Physiol. 48, 765-769). At least six different genes coding for starch synthases are expressed in maize, although previously it was not known which of these is responsible for the SSII activity peak. The enzyme activity in the SSII peak was neutralized to a large extent by antibodies raised against the product of the Du1 gene, but was not affected by antibodies specific for the other highly expressed soluble starch synthase, zSSI, or for the zSSIIa or zSSIIb isoforms. These data provide direct evidence that Du1 codes for the starch synthase responsible for the SSII activity peak. This starch synthase was purified approximately 350-fold from endosperm extracts. The following enzymatic properties of the SSII activity were determined: temperature optimum, thermostability, pH effects, K(m) for different glucan primers and the glucosyl unit donor ADPGlc, V(max) using various primers, and stimulation by citrate. These properties were compared to those of zSSI purified over 1600-fold from maize endosperm by a parallel procedure. The major differences between the two enzymes were that the SSII activity displayed higher K(m) values for ADPGlc, a distinct temperature range for maximal activity, and different relative activities toward specific exogenous substrates. The purified SSI and SSII activities both were shown to be capable of elongating maltooligosaccharide primers in vitro.

Prevention of allergic eye disease by treatment with IL-1 receptor antagonist.

PURPOSE: To determine the impact of interleukin-1 (IL-1) inhibition using IL-1 receptor antagonist (IL-1Ra) in a mouse model of allergic eye disease. METHODS: A/J mice sensitized and challenged with cat dander in the eye were treated with topical IL-1Ra or vehicle alone. Control mice were treated with IL-1Ra or vehicle but sensitized and challenged with phosphate-buffered saline alone. Immediately after the final allergen challenge, the mice were observed for behavioral changes and assessed for lid injection and chemosis. The animals were then killed, eyes and attached lids were removed for either RNA extraction or histology, and draining lymph nodes were removed for either RNA extraction or in vitro stimulation assays. Differences in chemokine message between experimental and control groups-were determined by RNase protection assays. RESULTS: Treatment with IL-1Ra in allergen-challenged animals significantly reduced allergen-induced changes in photosensitivity (60%, P = 0.0002), chemosis (50%, P = 0.0151), and injection (86.7%, P = 0.0068) compared with vehicle-treated controls. Interleukin-1Ra reduced the number of degranulated mast cells and caused a significant reduction in the number of eosinophils infiltrating the conjunctival matrix (P<0.001) after allergen challenge. Examination of chemokine mRNA taken from the conjunctiva and draining lymph nodes by RNase protection assay showed a profound decrease in the production of a number of C-C chemokines. CONCLUSIONS: These findings suggest that IL-1Ra is suppressing allergic eye disease by a down-modulation of the recruitment of eosinophils and other inflammatory cells essential for the immunopathogenesis of ocular atopy.

Measuring accumulated health-related benefits of exercise participation for older adults: the Vitality Plus Scale.

BACKGROUND: Existing measures fail to capture the perceived benefits attributed to exercise participation by older adults themselves. Noticeable improvements in sleep, energy level, bodily aches and pains, constipation, and other psychophysical aspects of "feeling good" may represent ongoing sources of motivation for continued participation. The Vitality Plus Scale (VPS) was developed to measure these potential health-related benefits of exercising. METHODS: The 10-item VPS was developed using an inductive approach, in collaboration with regularly exercising older adults and their instructors. Multiple samples of exercises and nonexercisers ranging in age from 40 to 94 were used to examine the reliability and validity of the new scale. RESULTS: The VPS showed good internal consistency and test-retest reliability over one week. Scores were able to discriminate on the basis of various indicators of health status and self-reported level of physical activity, and were related to two measures of functional mobility. Convergence was found with several subscales of the SF-36, whereas low correlations emerged with a measure of episode-specific sensations. Responsiveness to change was found with various types of exercise for individuals with low to moderate scores prior to participation. CONCLUSIONS: Improvements in sleep, energy level, mood, and generally feeling good appear to be the most noticeable benefits of exercising for many adults. These associations are reinforced by sustained exercise participation. Capturing these interrelated psychophysical constructs in a single, short measure will enable exercise researchers and instructors to measure incremental improvements previously reported only anecdotally.

Depletion of CD34+ CD4+ cells in bone marrow from HIV-1-infected individuals.

Pancytopenia as a consequence of bone marrow abnormalities is commonly seen in HIV-infected individuals. To examine the effect that HIV-1 has on hematopoietic cells, we compared hematopoietic properties of bone marrow samples from HTV+ patients at various stages of disease with bone marrow samples from uninfected donors. While the absolute number of recovered CD34+ cells and the cloning efficiency of these cells did not differ significantly in HIV+ donors, the percentage of CD34+ CD4+ cells was significantly depleted in late-stage HIV+ patients. We observed a direct correlation between the numbers of CD34+ CD4+ cells in the bone marrow and the peripheral CD4 count. Further characterization of the CD34+ CD4+ subpopulation demonstrated that these cells expressed lower levels of HLA-DR on their surface compared with CD34+ CD4- cells, suggesting an immature phenotype. We also found evidence for expression of HIV-1 coreceptors CXCR-4 and CKR-5 message and protein in CD34+ bone marrow cells. While this finding suggested that hematopoietic cells might be susceptible to HIV infection at an early stage of maturation, thus affecting different cell lineages as they matured, we did not find any evidence for infection of HIV in these cells. These data suggest that HIV affects early hematopoietic progenitor cells either directly or indirectly, and in particular CD34+ CD4+ cells. This finding has important implications for disease pathogenesis and for application of gene therapy approaches that use CD34+ hematopoietic cells.

Identification of the soluble starch synthase activities of maize endosperm.

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.

Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize.

This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and su1-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.

Control of Saccharomyces cerevisiae filamentous growth by cyclin-dependent kinase Cdc28.

The ascomycete Saccharomyces cerevisiae exhibits alternative vegetative growth states referred to as the yeast form and the filamentous form, and it switches between the two morphologies depending on specific environmental signals. To identify molecules involved in control of morphologic differentiation, this study characterized mutant S. cerevisiae strains that exhibit filamentous growth in the absence of the normal external signals. A specific amino acid substitution in the cyclin-dependent protein kinase Cdc28 was found to cause constitutive expression of most filamentous growth characteristics. These effects include specifically modified cell polarity characteristics in addition to the defined shape and division cycle alterations typical of the filamentous form. Several other mutations affecting Cdc28 function also had specific effects on filamentous growth. Constitutive filamentous growth resulting from deletion of the protein kinase Elm1 was prevented by modification of Cdc28 such that it could not be phosphorylated on tyrosine residue 19. In addition, various mutations affecting Hsl1 or Swe1, known or presumed components of a protein kinase cascade that mediates Cdc28 phosphorylation on Y19, either prevented or enhanced filamentous growth. The data suggest that a protein kinase cascade involving Elm1, Hsl1, and Swe1 can modulate Cdc28 activity and that Cdc28 in turn exerts global effects that cause filamentous growth.