Recruitment outcomes, challenges and lessons learned: the Healthy Communities Study.

BACKGROUND: The Healthy Communities Study (HCS) was a national study of community programs and policies that aimed to address childhood obesity; it necessitated recruitment of a large sample of children from communities throughout the United States. OBJECTIVE: The HCS aimed to complete visits with an average of 45 children and 12 key informants from at least 120 communities, diverse with respect to region of the country, urbanicity, socioeconomic status, race, ethnicity and intensity of community programs and policies that aim to address childhood obesity. METHODS: Purchased address lists were utilized to select households for recruitment during Wave 1 of the study, and recruitment of families through schools was employed for Wave 2. RESULTS: The HCS successfully obtained approval from 149 school districts and 478 schools in 130 communities, recruited 5138 families, and interviewed 1421 key informants to allow for characterization of overall intensity of obesity prevention/treatment efforts in each community. CONCLUSIONS: Lessons learned are presented. Future studies should plan for inclusion of the following in development of recruitment strategies: literature review, formative research, pilot testing, and ongoing monitoring and adjustment.

The longitudinal relationship between community programmes and policies to prevent childhood obesity and BMI in children: the Healthy Communities Study.

BACKGROUND: Although a national epidemic of childhood obesity is apparent, how community-based programmes and policies (CPPs) affect this outcome is not well understood. OBJECTIVES: This study examined the longitudinal relationship between the intensity of CPPs in 130 communities over 10 years and body mass index (BMI) of resident children. We also examined whether these relationships differ by key family or community characteristics. METHODS: Five thousand one hundred thirty-eight children in grades K-8 were recruited through 436 schools located within 130 diverse US communities. Measures of height, weight, nutrition, physical activity and behavioural and demographic family characteristics were obtained during in-home visits. A subsample of families consented to medical record review; these weight and height measures were used to calculate BMI over time for 3227 children. A total of 9681 CPPs were reported during structured interviews of 1421 community key informants, and used to calculate a time series of CPP intensity scores within each community over the previous decade. Linear mixed effect models were used to assess longitudinal relationships between childhood BMI and CPP intensity. RESULTS: An average BMI difference of 1.4 kg/m2 (p-value < 0.01) was observed between communities with the highest and lowest observed CPP intensity scores, after adjusting for community and child level covariates. BMI/CPP relationships differed significantly by child grade, race/ethnicity, family income and parental education; as well as community-level race/ethnicity. CONCLUSIONS: These results indicate that, over time, more intense CPP interventions are related to lower childhood BMI, and that there are disparities in this association by sociodemographic characteristics of families and communities.

Monoclonal antibody (EGR/G49) reactive with the epidermal growth factor receptor of A431 cells recognizes the blood group ALeb and ALey structures.

The carbohydrate specificity of the monoclonal antibody EGR/G49, raised against the epidermal growth factor (EGF) receptor of A431 cells, has been investigated by assessing its interactions with glycoproteins and erythrocytes derived from individuals of known blood group ABH, Lewis and secretor types, and by inhibition of binding assays using structurally defined oligosaccharides. The results indicate that this antibody reacts with the difucosylated blood group structures ALeb and ALey: (formula; see text) This antibody differs from the previously described anti-EGF receptor antibody. TL5, which is directed at the terminal blood group A trisaccharide structure and reacts poorly with the ALeb/Ley structures. Since both antibodies were selected for their reactivities with the receptor for EGF, their specificities provide evidence for the presence of both the mono- and difucosylated blood group A structures on the receptor glycoprotein. These antibodies will be invaluable in the studies of the distribution and the roles of blood group related carbohydrate structures in the organisation and function of the EGF and other receptor systems.

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

p53 protein expression in breast carcinomas. Comparative study with the wild type p53 induced proteins mdm2 and p21/waf1.

The aim was to investigate the pattern of expression of p53 protein and two wild-type (wt) p53-induced proteins (mdm2 and p21/waf1), as an indirect way of assessing p53 gene status in breast carcinomas. Formalin-fixed paraffin embedded tissue from 102 cases of breast carcinomas comprising mostly ductal carcinomas (88 cases) was stained by immunohistochemistry for p53, mdm2 and p21/waf1 proteins. We found p53, mdm2 and waf1/p21 protein expression in 33/102, 20/102 and 38/102 breast carcinomas, respectively. Parallel p53/mdm2 protein expression was found in 9 cases. Five were also p21/waf1 positive. Discordant p53+/ mdm2-protein expression was found in 24 cases. Nine were p21/waf1 positive and the remaining fifteen were p21/waf1 negative. The patterns mdm2+/p53-/p21- and p21+/p53-(+)/mdm2- were found in 6 and 20 cases, respectively. Parallel p53/mdm2/p21 protein expression may represent breast carcinomas with wt p53 gene since mdm2 and p21 proteins are inducible by wt p53 gene. In these cases p53 protein expression may be due to stabilisation to mdm2 protein. This could be important in the pathogenesis of these cases since mdm2 may deregulate the p53-dependent growth suppressive pathway. Discordant p53+/mdm2-/p21- protein expression may represent breast carcinomas with p53 gene mutations unable to activate expression of mdm2 and p21 proteins. Breast carcinomas with p53+/mdm2/p21+ protein expression may have either wt p53 with deregulated mdm2 gene expression or mutated p53 gene with p53-independent p21 expression. Cases with only mdm2 expression may represent tumours with mdm2 gene amplification or overexpression and cases with only p21 expression may reflect p53-independent regulation of p21 protein.

Inhibition of the aliphatic amidase from Pseudomonas aeruginosa by urea and related compounds.

The time-dependent inhibition of amidase from Pseudomonas aeruginosa strain AI 3 by urea, hydroxyurea and cyanate displayed saturation kinetics fitting a model for the reaction sequence in which formation of a complex in a reversible step was followed by an irreversible step. Altered amidases from mutant strains AIU 1N and OUCH 4, selected for their resistance to inhibition of growth by urea and hydroxyurea respectively, had altered kinetic constants for inhibition indicating reduced binding capacity for the inhibitors. The substrate acetamide protected AI 3 amidase against inhibition by urea,.and altered Ki values for inhibition of the mutant amidases were paralleled by alterations in Km values for acetamide indicating that urea acted at the active site. Inhibition of AI 3 amidase involved the binding of one molecule of urea per molecule of enzyme. Urea inhibited amidase slowly regained activity at pH 7.2 through release of urea.

Adaptation to phenylacetamide as a growth substrate by an acetanilide-utilizing mutant of Pseudomonas aeruginosa.

Mutants able to utilize phenylacetamide as sole nitrogen source were isolated from the acetanilide (N-phenylacetamide) - utilizing Pseudomonas aeruginosa mutant strain A13 and from its parent strain L 10. Growth properties of the mutants (Ph strains) on amide media and the physicochemical properties of their amidases in cell free extracts indicated that their phenylacetamidase activities were attributable to alterations in their amidases. Differences in amide hydrolase specificities between the AI3- and the L 10-Ph mutants were observed. The AI3 group had a high level of activity towards 4-nitrophenylacetamide, activity towards phenylacetyl-4-nitroaniline but, unlike strain AI3, no activity towards acetyl-4-nitroaniline; the L 10 group had a low activity towards 4-nitrophenylacetamide, no activity towards phenylacetyl-4-nitroaniline but retained the low level of activity towards acetyl-4-nitroaniline exhibited by strain L 10. Confirmation of the association between these altered specificities and alterations in amidases was obtained from analysis of the properties of phenylacetamidases purified from an AI3-Ph mutant (pH 5) and an L 10-Ph mutant (Ph14). The original mutation in the amidase gene of strain AI3 appeared responsible for the differences between the two groups of Ph mutants and the binding interactions with acetanilide that it determined were eliminated in AI3-Ph mutants.

Isotope-exchange evidence that glucose 6-phosphate inhibits rat-muscle hexokinase II at an allosteric site.

The flux ratio for hexokinase type II from rat muscle, i.e. the rate of conversion of glucose 6-phosphate molecules into ATP molecules divided by the simultaneous rate of conversion of glucose 6-phosphate molecules into glucose molecules, increases with the MgATP concentration but is independent of the glucose concentration. This behaviour requires that glucose must bind before MgATP when the reaction is proceeding in the normal physiological direction, i.e. phosphorylation of glucose. Although at low non-inhibitory glucose 6-phosphate concentrations the flux ratio increases linearly with the MgATP concentration, the dependence becomes non-linear, with a slope that increases with the MgATP concentration, at glucose 6-phosphate concentrations above 1 mM. This behaviour does not permit glucose 6-phosphate to act only as a normal product inhibitor. Instead, it seems to require glucose 6-phosphate to act as an allosteric inhibitor and for a second site for binding of MgATP to exist. Measurements of the flux from ATP to glucose 6-phosphate and to ADP showed no dependence of the flux ratio on the concentrations of either glucose 6-phosphate or ADP. This result does not permit the order of product-release steps in this direction to be determined, but shows that the second product is released virtually instantaneously after first.

Allosteric character of the inhibition of rat-muscle hexokinase B by glucose 6-phosphate.

We previously provided evidence from isotope-exchange measurements under non-equilibrium conditions that hexokinase B from rat muscle follows a compulsory-order mechanism with glucose binding before MgATP, and with both glucose 6-phosphate and MgATP capable of binding allosterically [Gregoriou, M., Trayer, I. P. & Cornish-Bowden, A. (1983) Eur. J. Biochem. 134, 283-288]. We have now re-examined this work in the light of recent criticisms [Ganson, N. J. & Fromm, H. J. (1985) J. Biol. Chem. 260, 12099-12105]. There is no difficulty in obtaining valid estimates of initial rates of isotope exchange when the equilibrium constant is unfavourable, if one uses highly radioactive reactants and low enzyme concentrations, as we did in the experiments we reported previously. However, our earlier suggestion that MgADP can be released within the inhibitory pathway, which was made for the sake of consistency with the catalytic pathway rather than because of any compelling experimental evidence, must be revised to avoid predicting that the rate must be zero in the absence of MgADP. Although our mechanism admits the possibility of substrate inhibition by MgATP, calculations show that there is no need for this to be observable under ordinary conditions. Indeed, with plausible values assumed for the kinetic constants one can calculate theoretical behaviour according to our model that closely resembles the experimental inhibition experiments that have been claimed as evidence against it.

An investigation of the role of Glu-842, Glu-844 and His-846 in the function of the cytoplasmic domain of the epidermal growth factor receptor.

Activation of several protein kinases is mediated, at least in part, by phosphorylation of conserved Thr or Tyr residues located in a variable loop region, near the active site. In certain kinases, this activation loop also controls access of peptide substrates to the active site. In the corresponding region of the epidermal growth factor (EGF) receptor, a potential phosphorylation site, Tyr-845, does not appear to have a major regulatory role. In order to find out whether this variable loop can modulate the peptide phosphorylation and self-phosphorylation activities of the EGF receptor kinase, we investigated the role of residues around Tyr-845, using site-directed mutagenesis. Multiple sequence alignment showed that residues Glu-842, Glu-844 and His-846 are conserved or nearly conserved in eight members of the EGF receptor family. Mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala were expressed in the baculovirus/insect cell system, purified to near-homogeneity and characterized with respect to their peptide phosphorylation and self-phosphorylation activities. All three mutants were active, and these changes did not affect ATP binding directly. However, all mutations increased the Km(app.) for peptide substrates and MnATP in peptide phosphorylation reactions. The Vmax. for the phosphorylation of peptide RREELQDDYEDD was unaltered, but the Vmax. for self-phosphorylation (with variable [MnATP]) decreased 4-, 2- and 7-fold for mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala respectively, compared with the wild-type. These results suggest that binding of this peptide restored an optimal conformation at the active site that might be impaired by the mutations. A study of the dependence of initial rates of self-phosphorylation on cytoplasmic domain concentration showed that the order of reaction increased with the progress of self-phosphorylation. Both pre-phosphorylation and high concentrations of ammonium sulphate restored maximal or near-maximal levels of self-phosphorylation in the mutants, possibly through compensating conformational changes. A plausible homology model, based on the cyclic AMP-dependent protein kinase catalytic subunit, accommodated the sequence Glu-841-Glu-Lys-Glu as an insertion in the peptide binding loop at the edge of the active site cleft. The model suggests that Glu-844 and His-846 may participate in H-bonding interactions, thus stabilizing the active site region, while Glu-842 does not appear to interact with regions of the catalytic core.

Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.

Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor.

Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI3. AI3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2. Compared with AI3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.

Isotope-exchange evidence for an ordered mechanism for rat-liver glucokinase, a monomeric cooperative enzyme.

The order of addition of substrates and release of products in the reaction catalyzed by rat-liver glucokinase has been studied by measurements of isotope exchange. Experiments at chemical equilibrium showed some degree of randomness, but steady-state experiments showed a predominantly ordered process with glucose binding first and glucose 6-phosphate released last. Experiments to trap binary complexes in the steady state demonstrated the existence of complexes of the enzyme with glucose and with glucose 6-phosphate but gave no evidence for the occurrence of corresponding complexes with ATP or ADP. Flux ratios measured in both the forward and reverse reactions provided a more rigorous and quantitative confirmation of these characteristics of the reaction. These observations support the interpretation of glucokinase cooperativity in terms of a "mnemonical" mechanism and conflict with an alternative interpretation in terms of a random addition of substrates.

Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells.

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion.

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.

Investigation of the expression of epidermal growth factor receptor and blood group A antigen in 110 human gliomas.

The presence of epidermal growth factor receptor (EGF-R) and blood group A antigen was studied immunohistochemically in a series of 110 malignant gliomas using monoclonal antibodies. Fifty-seven percent of the tumours strongly expressed EGF-R on the malignant cells. Although blood group A antigen is present on EGF-R of A431 cells (a cell line derived from a human epidermoid carcinoma), in gliomas it was found only on vascular endothelial cells of tumours from blood group A patients. The results suggest that the EGF-R present in gliomas differs from that in A431 cells in the type or amount of the carbohydrate chains. This is in contrast to previous reports which have suggested that A antigen is present on EGF-R in gliomas. This has relevance in the choice of monoclonal antibodies used to study the EGF-R, as those directed against the A antigen component of the A431 cell EGF-R will not recognize EGF-R elsewhere and may cause normal blood group A antigen to be mistaken for EGF-R.

The calpain cleavage sites in the epidermal growth factor receptor kinase domain.

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N- or C-termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N-terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996-Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxta-membrane domain, C-terminal to the regulatory Thr654. We purified an active 42-kDa fragment generated by calpain proteolysis between Leu659-Gln660 in the juxta-membrane domain, and in the hinge region. A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly-Xaa-Gly-Xaa-Xaa-Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down-regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.

Epidermal growth factor receptor expression in 72 meningiomas.

The expression of epidermal growth factor receptor (EGF-R) was studied immunohistochemically in 72 meningiomas using two monoclonal antibodies with specificities to protein and carbohydrate components, respectively, of the external domain of the EGF-R. One third of the tumors had cytoplasmic and membrane positivity with the protein-specific antibody but in none were there positive tumor cells with the carbohydrate-specific antibody which recognizes the blood group A antigen. There was no difference in EGF-R expression between typical and aggressive meningiomas. No evidence was found to support previous reports of specific EGF-R immunoreactivity in the vascular endothelial cells of meningiomas. The authors believe this discrepancy to be due to detection of normal blood group A antigen attached to endothelial cells in patients of blood group A or AB. This occurs because many monoclonal anti-EGF-R antibodies are specific for A antigen which is found on the EGF-R of A431 cells but has not been reported on EGF-R elsewhere.