The effectiveness of local child weight management programmes: an audit study.

BACKGROUND: Many different child weight management programmes exist, with varying degrees of evaluation to provide evidence of their success. The purpose of this research was to use a standardized approach to audit the effectiveness of weight management intervention programmes in the West Midlands region of the UK, specifically to assess the benefits to participating children in terms of health improvement and behaviour change. METHODS: An audit of seven family-based intervention programmes currently in place in the West Midlands. Programmes were audited against the Standard Evaluation Framework. RESULTS: The programmes provided a partial data set relating to a change in weight from the baseline to the end of the programme; none of the programmes provided all of the measures indicated by the Standard Evaluation Form as being essential for evaluation. Weight change ranged from an increase in group mean of 0.4 kg to a decrease of 0.9 kg. Body Mass Index SD decreased by 0.1-0.2 points in four programmes and remained unchanged in two programmes. Four programmes collected long-term follow-up data at 6 months. This was often limited because of participant dropout. Improvement in diet and exercise were reported by participants in all programmes which measured these behaviours. CONCLUSIONS: Ongoing evaluation of all programmes, using a standard approach, is essential in order to improve the evidence base and support future commissioning.

The impact of school-day variation in weight and height on National Child Measurement Programme body mass index-determined weight category in Year 6 children.

BACKGROUND: In England, the National Child Measurement Programme (NCMP) annually measures the weight and height of Year 6 schoolchildren (age 10-11 years). While measurement protocols are defined, the time of measurement within the school day is not. This study examined the impact of school-day variation in weight and height on NCMP body mass index (BMI)-determined weight category in Year 6 children. METHODS: Standing height and weight were measured in morning and afternoon sessions in 74 children, boys (n= 34; height: 141.16 ± 7.45 cm; weight: 36.48 ± 9.46 kg, BMI: 18.19 ± 3.98 kg/m(2) ) and girls (n= 40; height: 144.58 ± 7.66 cm; weight: 42.25 ± 11.29 kg; BMI: 19.97 ± 3.98 kg/m(2) ) aged 11 ± 0.3 years. RESULTS: In the whole sample, height decreased (Mean =-0.51 cm, 95% CI: -0.39 to -0.64 cm, P= 0.01), weight did not change (Mdn = 36.40 to 36.35, P= 0.09) and BMI increased (Mdn = 18.04 to 18.13, P= 0.01). In girls weight increased (Mdn = 41.40 to 41.60, P= 0.01). BMI percentile increased (Mdn = 57th to 59.5th centile, P= 0.01). One girl increased in BMI category from morning to afternoon according to the clinical cut-offs (≤2nd, >91st and >98th) and three girls increased BMI category according to the population monitoring cut-offs (≤2nd, ≥85th, ≥95th). CONCLUSIONS: School-day variation in height (and in girls alone, weight) impact upon increased BMI and BMI percentile in afternoon versus morning measurements in Year 6 children. Although not reaching statistical significance, resultant variation in categorization at the individual level may lead to unwarranted follow-up procedures being initiated. Further research with larger samples is required to further explore the impact of daily variability in height and weight upon both clinical and population monitoring BMI-determined weight status categorization in the NCMP.

Magnetic resonance imaging of abdominal adiposity in a large cohort of British children.

OBJECTIVE: To describe abdominal adipose tissue distribution in a large sample of contemporary British children; to determine the influence of gender, stage of maturation and body mass index (BMI) on abdominal adipose tissue distribution; and to compare the ability of BMI and waist circumference to predict abdominal adipose tissue. SUBJECTS AND METHODS: A total of 74 boys (mean age 13.4+/-0.4 years) and 96 girls (mean age 13.5+/-0.5 years) were selected from volunteer children enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC). Height, weight and waist circumference were measured and BMI calculated. Stage of sexual maturation was available for 113 children using a self-report questionnaire based on Tanner's criteria. Magnetic resonance imaging was used to assess subcutaneous abdominal adipose tissue (SAAT) and intra-abdominal adipose tissue (IAAT) volumes and patterning. RESULTS: Boys had lower levels of IAAT (P=0.036) and SAAT (P=0.003) than girls. IAAT and SAAT were higher in overweight and obese boys and girls when compared with normal weight children (P<0.0001). This pattern was also reflected in waist circumference groups. Boys had higher IAAT/SAAT ratios than girls, indicating proportionately more adipose tissue deposited intra-abdominally (P=0.002). However, both boys and girls deposited less than 10% of their abdominal fat as internal adipose tissue. WC predicted 67.4% of the variance in IAAT (P<0.001), and BMI predicted 84.8% of the variance in SAAT (P<0.001). However, BMI as the best single predictor explained only 8.4% of the variance in the IAAT/SAAT ratio (P<0.001). CONCLUSIONS: At this age and stage of sexual maturation, the amount of IAAT remains relatively small. WC and BMI offer a feasible alternative to the MRI estimation of IAAT and SAAT, respectively, in a population-based sample of boys and girls. International Journal of Obesity (2008) 32, 91-99; doi:10.1038/sj.ijo.0803780; published online 27 November 2007.

Crossvalidation of anthropometry against magnetic resonance imaging for the assessment of visceral and subcutaneous adipose tissue in children.

BACKGROUND: The study of the relationship between anthropometry and visceral adipose tissue (VAT) is of great interest because VAT is associated with many risk factors for noncommunicable diseases and anthropometry is easy to perform in clinical practice. The studies hitherto available for children have, however, been performed on small sample sizes. DESIGN: Pooling of the data of studies published from 1992 to 2004 as indexed on Medline. AIMS: To assess the relationship between anthropometry and VAT and subcutaneous adipose tissue (SAT) as measured by magnetic resonance imaging (MRI) in children and to analyze the effect of age, gender, pubertal status and ethnicity. SUBJECTS AND METHODS: Eligible subjects were 7-16 year-old, with availability of VAT and SAT, gender, ethnicity, body mass index (BMI) and waist circumference (WC). A total of 497 subjects were collected from seven different investigators and 407 of them (178 Caucasians and 229 Hispanics) were analyzed. RESULTS: Despite ethnic differences in MRI data, BMI, WC and age, no difference in VAT was found between Caucasians and Hispanics after correction for SAT and BMI. Univariate regression analysis identified WC as the best single predictor of VAT (64.8% of variance) and BMI of SAT (88.9% of variance). The contribution of ethnicity and gender to the unexplained variance of the VAT-WC relationship was low (< or =3%) but significant (P < or =0.002). The different laboratories explained a low (< or =4.8%) but significant (P < 0.0001) portion of the unexplained variance of the VAT-WC and SAT-BMI relationships. Prediction equations for VAT (VAT (cm(2)) = 1.1 x WC (cm)-52.9) and SAT (SAT (cm(2)) = 23.2 x BMI (kg/m(2))-329) were developed on a randomly chosen half of the population and crossvalidated in the remaining half. The pure error of the estimate was 13 cm(2) for VAT and 57 cm(2) for SAT. CONCLUSIONS: WC can be considered a good predictor of VAT as well as BMI of SAT. The importance of ethnicity and gender on VAT estimation is not negligible.

Assessment of abdominal fat development in young adolescents using magnetic resonance imaging.

OBJECTIVE: To determine the patterns of change and the best anthropometric indicators of intra-abdominal fat deposition in young adolescents from ages 11-13 y. SUBJECTS: Subjects were 25 boys (mean age 13.7 +/- 0.32 y) and 17 girls (mean age of 13.7 +/- 0.23 y) who had taken part in a similar study 2 y earlier at ages 11.5 +/- 0.33 y and 11.5 +/- 0.27 y, respectively. METHODS: Intra-abdominal (IA) and subcutaneous adipose (SA) tissue areas and IA/SA ratio were determined through four tranverse magnetic resonance imaging scans on two occasions. Differences were investigated using t-tests and ANOVA. Skinfolds, girths and circumferences, body mass index and hydrostatic weighing were also recorded. Pearson correlation coefficients and regression equations were calculated to determine the best anthropometric indicators of intra-abdominal fat deposition. RESULTS: Intra-abdominal fat and subcutaneous fat areas had significantly increased in boys and girls by the second measure. Boys had deposited greater amounts of fat in intra-abdominal depots so that their intra-abdominal/subcutaneous ratio had increased significantly from 0.31 to 0.39. This had reduced in girls from 0.39 to 0.35. However, patterns of change were variable within sexes. Truncal skinfold sites (r = 0.54-0.70) emerged as the best field indicators of intra-abdominal fat deposition. CONCLUSIONS: Patterns of intra-abdominal and subcutaneous fat distribution are identifiable in pubescent children using magnetic resonance imaging. An acceptable indication is provided by truncal skinfolds.

The biologic response to laser thermal modification in an in vivo sheep model.

The purpose of this study was to evaluate the effect of nonablative laser energy on mechanical, histologic, ultrastructural, and biochemical properties of joint capsular tissue in an in vivo sheep model. Femoropatellar joint capsule was treated with the holmium:yttrium-aluminum-garnet laser via an arthroscope, and tissues were harvested immediately after surgery, or at 3, 7, 14, 30, 60, 90, and 180 days after surgery (n = 8/group). Laser treatment caused significant decreases in tissue stiffness from 0 to 7 days after surgery, then stiffness gradually increased after 14 days. Tissue strength was lowest 3 days after laser treatment. Histologic examination revealed immediate collagen hyalinization and cell necrosis, followed by active cellular response characterized by extensive fibroblast migration and capillary sprouting. Tissue appeared to be normal histologically 60 days after surgery; however, collagen fibrils remained uniformly small. This study showed an active tissue response secondary to thermal modification with concomitant recovery of mechanical properties by 30 days after surgery. Whether the shrinkage or joint stability was maintained with time remains to be evaluated. To clarify the advantages and disadvantages of this technique, a carefully controlled clinical trial with long term followup should be performed.

The mechanism of joint capsule thermal modification in an in-vitro sheep model.

The purpose of this study was to understand the mechanism responsible for joint capsule shrinkage after nonablative laser application in an in-vitro sheep model. Femoropatellar joint capsular tissue specimens harvested from 20 adult sheep were treated with one of three power settings of a holmium:yttrium-aluminum-garnet laser or served as a control. Laser treatment significantly shortened the tissue and decreased tissue stiffness in all three laser groups, whereas failure strength was not altered significantly by laser treatment. Transmission electron microscopic examination showed swollen collagen fibrils and loss of membrane integrity of fibroblasts. A thermometric study revealed nonablative laser energy caused tissue temperature to rise in the range of 64 degrees C to 100 degrees C. Electrophoresis after trypsin digestion of the tissue revealed significant loss of distinct alpha bands of Type I collagen in laser treated samples, whereas alpha bands were present in laser treated tissue without trypsin digestion. The results of this study support the concept that the primary mechanism responsible for the effect of nonablative laser energy is thermal denaturation of collagen in joint capsular tissue associated with unwinding of the triple helical structure of the collagen molecule.

Agonist-induced desensitization of A2B adenosine receptors.

Agonist-induced desensitization has been described for the A1, A2A, and A3 adenosine receptor subtypes of the G protein-coupled receptor superfamily. Desensitization of the fourth adenosine receptor subtype, the A2B adenosine receptor (A(2B)R), has not been studied extensively. We sought to determine whether the A(2B)R is subject to agonist-induced desensitization. COS 7 cells, which exhibit endogenous expression of the A(2B)R, and transfected CHO cells, which stably express a modified rat A(2B)R bearing a 5' FLAG epitope tag, were studied. Cyclic AMP (cAMP) responsiveness to an acute challenge was measured after pretreating (desensitizing) cells with the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). Incubation with NECA resulted in hyporesponsiveness to acute agonist challenge in both COS 7 and transfected CHO cells. Desensitized cells exhibited restoration of cAMP responses after recovery for 24 hr in growth medium. Choleratoxin-induced cAMP responses were preserved in desensitized cells, and high concentrations of NECA were unable to overcome the desensitization. Membrane levels of the epitope-tagged A(2B)R were assessed by western blot in transiently transfected COS 7 cells. The expression of epitope-tagged A(2B)Rs was not different between control and desensitized cells. In northern blot analysis, levels of endogenous A(2B)R mRNA were similar in control and desensitized COS 7 cells. We conclude that the A(2B)R is subject to agonist-induced desensitization with preserved expression of A(2B)R mRNA and protein. Uncoupling of the A2B adenosine receptor from the G protein complex may contribute to the mechanism of desensitization.

Fibronectin fibrillogenesis involves the heparin II binding domain of fibronectin.

Fibronectin matrix assembly is thought to involve binding interactions between the amino-terminal I1-5 repeats and the first type III repeat (III1). Here we report that a third site, located within the III12-14 repeats of the carboxyl-terminal heparin II domain of fibronectin, is also involved in fibrillogenesis. Heparin II fragments inhibited fibril formation and binding of 125I-labeled fibronectin and/or 70-kDa fragments to the cell surface, deoxycholate-insoluble matrix, and adsorbed 160-kDa cell adhesion fragments of fibronectin. The inhibitory effects of heparin II fragments were as large or up to 20 times larger than those of a 44-kDa fibronectin fragment containing the III1 repeat. Under physiological conditions, amino-terminal fragments of fibronectin containing the I1-5 repeats interacted preferentially with proteolytically derived heparin II fragments and a recombinant III12-14 peptide both in solution and in solid phase, indicating that matrix assembly may involve direct interactions between I1-5 and III12-14 repeats. Interactions between the I1-5 repeats and 160-kDa fragments containing the III12-14 and III1 repeats could be inhibited by >/= 90% by either an anti-III13-14 monoclonal antibody (mAb) (IST-2) or an anti-III1 mAb (9D2), suggesting that cooperative interactions between III12-14 and III1 repeats may also promote binding of the I1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I1-5 binding sites, independent of the III1 and III12-14 repeats, may be involved in fibrillogenesis.

Regulation of mu opioid receptor mRNA levels by activation of protein kinase C in human SH-SY5Y neuroblastoma cells.

BACKGROUND: The mu opioid receptor (MuOR) is a member of the superfamily of G protein-coupled receptors that mediates the analgesic actions of endogenous opioid peptides and the narcotic alkaloid derivatives of morphine. Activation and translocation of protein kinase C (PKC) by N-methyl-D-aspartate receptor stimulation correlates with resistance to opioid drugs in experimental states of neuropathic pain, but the cellular mechanisms of resistance have not been identified. One possibility is that PKC activation regulates MuOR mRNA expression and thus the ability to generate functional receptors. Using a human neuroblastoma cell line, the authors tested the hypothesis that phorbol ester activation of PKC regulates MuOR mRNA levels. METHODS: SH-SY5Y cells were maintained in a continuous monolayer culture and treated with phorbol esters or other agents before extraction of total cellular RNA. Slot-blot hybridization was used to measure the level of MuOR mRNA using 32P-labeled MuOR cDNA probes under high-stringency conditions. Autoradiograms were analyzed by scanning and densitometry. RESULTS: MuOR mRNA levels decreased in a dose- and time-dependent manner after tetradecanoyl phorbol acetate (TPA) was administered to activate PKC. The nadir, a level of approximately 50% of control, was at 2-8 h, followed by gradual recovery. The actions of TPA were blocked by pretreatment with the selective PKC inhibitor bisindolylmaleimide, but not by inhibition of protein synthesis with cycloheximide or anisomycin. The combination of TPA treatment and transcription inhibition with actinomycin D was associated with a transient increase in MuOR mRNA. CONCLUSIONS: Mu opioid receptor mRNA levels are regulated by activation of PKC in a neuronal model. Protein kinase C effects which decrease MuOR mRNA levels appear largely independent of new protein synthesis, and cytotoxicity does not account for the findings. Plasticity of MuOR gene expression may contribute to variations in clinical responses to opioid analgesics in clinical states such as neuropathic pain.

High-resolution cryo-scanning electron microscopy study of the macromolecular structure of fibronectin fibrils.

High-resolution cryo-scanning electron microscopy was used to examine fibronectin fibrils formed in culture by human skin fibroblasts and in a cell-free system by denaturing soluble plasma fibronectin with guanidine. These studies indicate that the conformation of fibrils formed in culture and in a cell-free system appeared to be similar and that fibronectin fibrils have at least two distinct structural conformations. Fibronectin fibrils can be very straight structures with smooth surfaces or highly nodular structures. The average diameter of the nodules in these fibrils is 12 nm. Both conformations can be seen within an individual fibril indicating that they are not different types of fibronectin fibrils but rather different conformational states. Immunolabeling studies with a monoclonal antibody, IST-2, to the heparin II binding domain of fibronectin revealed that the epitope was buried in highly smooth fibrils, but it was readily exposed in nodular fibrils. We propose, therefore, that fibronectin fibrils are highly flexible structures and, depending on the conformation of the fibril, certain epitopes on the surface may be buried or exposed.

Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by Bmp1, a mammalian gene related to Drosophila tolloid.

The mouse bone morphogenetic protein1 (Bmp1) gene encodes a secreted astacin metalloprotease that cleaves the COOH-propeptide of procollagen I, II and III. BMP-1 is also related to the product of the Drosophila patterning gene, tolloid (tld), which enhances the activity of the TGFbeta-related growth factor Decapentaplegic and promotes development of the dorsalmost amnioserosa. We have disrupted the mouse Bmp1 gene by deleting DNA sequences encoding the active site of the astacin-like protease domain common to all splice variants. Homozygous mutant embryos appear to have a normal skeleton, apart from reduced ossification of certain skull bones. However, they have a persistent herniation of the gut in the umbilical region and do not survive beyond birth. Analysis of the amnion of homozygous mutant embryos reveals the absence of the fold that normally tightly encloses the physiological hernia of the gut. At the electron microscopic level, the extracellular matrix of the amnion contains collagen fibrils with an abnormal morphology, consistent with the incorporation of partially processed procollagen molecules. Metabolical labelling and immunofluorescence studies also reveal abnormal processing and deposition of procollagen by homozygous mutant fibroblasts in culture.

Maturation of collagen fibrils in the corneal stroma results in masking of tyrosine-rich region of type V procollagen.

PURPOSE: To determine the molecular form of type V procollagen in collagen fibrils in mammalian corneal stromas. METHODS: The presence of the tyrosine-rich region in the NH2-propeptide of type V procollagen in collagen fibrils was examined in human, bovine, and mouse corneas and human corneal fibroblast cultures by immunofluorescence microscopy and immunoblot analysis using a polyclonal antibody specific for this region. The antibody was generated using a glutathione S-transferase-fusion peptide. RESULTS: The tyrosine-rich region was detected readily in frozen sections of 5- to 6-month-old mouse corneal stromas without the need for any unmasking techniques, indicating that this domain is exposed on the surface of striated collagen fibrils. In contrast, frozen sections of adult human and bovine corneas did not label with the polyclonal sera to the tyrosine-rich region. Immunoblot analysis of bacterial collagenase digests of human and bovine corneas, however, indicated that peptide fragments containing the tyrosine-rich region of type V procollagen and of the expected molecular weight of 70 to 85 kDa were present. Further immunofluorescence microscopic studies and immunoblot analysis of mouse corneas at different ages and of collagen fibrils formed in human corneal fibroblast cultures over time indicated that, initially, the tyrosine-rich region of type V procollagen could be detected in all these collagen fibrils; however, as the age of the mouse and the culture increased, the ability to detect this region decreased. CONCLUSIONS: These results suggest that, in vivo, the tyrosine-rich region of type V procollagen is retained on type V procollagen molecules within mammalian collagen fibrils from corneal stromas and that this region becomes masked as collagen fibrils mature or the species ages.

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts.

Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the transfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a useful in vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell's lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid.

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.

Substrate-specific binding of the amino terminus of fibronectin to an integrin complex in focal adhesions.

The assembly of fibronectin fibrils involves the amino-terminal and cell adhesion domains of fibronectin as well as alpha 5 beta 1 integrins. Efficient binding of biotinylated or radioiodinated 70-kDa amino-terminal fragments occurred only if fibroblasts were plated on fibronectin or on 180- or 85-kDa cell adhesion fragments of fibronectin. On an 11.5-kDa fragment of fibronectin that included the Arg-Gly-Asp (RGD) sequence, but not the synergy site, binding was reduced 50-fold. Conformation of the 180-kDa fragment was important for direct binding interactions with the amino terminus of fibronectin. No binding was seen if cells were plated on type I collagen, vitronectin, RGD peptides or antibodies to alpha 5 beta 1 integrins. High affinity interactions between invasin and alpha 5 beta 1 integrin promoted low levels of binding. Monoclonal antibodies that blocked the function of either the RGD or the synergy site inhibited binding of 125I-labeled 70-kDa fragments to cells by approximately 60%. By fluorescence and interference reflection microscopy, biotinylated 70-kDa fragments were shown to co-localize with alpha 5 beta 1 integrins in focal adhesions. We propose that cell-mediated binding of the amino terminus of fibronectin involves interactions with both fibronectin and its alpha 5 beta 1 integrin receptor in an activated complex.

Fibronectin binding site in type I collagen regulates fibronectin fibril formation.

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.

Prediction and measurement of frame size in young adult males.

The assessment of frame size is a problematic and ambiguous area. The purpose of this study was to assess the level of agreement between various techniques of assessing frame size in a group of 27 healthy and active men aged 18-24 years, and also to assess which anthropometric variables were best associated with a measure of actual frame size (AFS) which is proposed in this study. Actual frame size was measured by the summation of a series of bone breadths, lengths and depths on a sub-sample of 17 men. The results of the study revealed substantial discordance between methods of assessing frame size. The variables which correlated most highly with AFS (P < 0.01) were body mass, ankle breadth, hand length and chest breadth, respectively. These variables were also positively correlated (P < 0.01) with fat-free mass (FFM), with no significant correlation with fat mass in either case. Of the various documented methods used to assess frame size, the 'HAT' technique, which incorporates biacromial and bitrochanteric breadths, was more highly correlated with AFS than both elbow breadth (currently used in height-weight insurance tables) and the height/wrist circumference index. The latter measure was not highly correlated with AFS, body mass and FFM in this study. It was concluded that ankle breadth and hand length may be better predictors of frame size in young men than other bone dimensions. In addition, the results of this preliminary investigation have substantiated the potential viability of an AFS model. Future research using this technique is recommended to determine true indicators of frame size in a larger and more heterogeneous population.

Arrangement of cellular fibronectin in noncollagenous fibrils in human fibroblast cultures.

The assembly of fibronectin into fibrils was examined by high-voltage immunoelectron microscopy in subconfluent cultures of ascorbate-treated human skin fibroblasts. Cells grown in the presence of ascorbic acid for 24, 48 or 72 h were labeled with Ist-9, a monoclonal antibody specific for the EIIIA site in fibronectin, and polyclonal antibodies to type I collagen. Cells were then labeled with goat anti-mouse IgG and goat anti-rabbit IgG coupled to 5 or 18 nm colloidal gold beads. Our results show that by 24 h, fibronectin is observed in fibrils in the extracellular matrix. The majority of fibronectin in fibrils does not co-localize with type I collagen. Morphometric analysis of the distance between EIIIA sites in fibronectin fibrils (less than 12 nm in diameter) show that the EIIIA sites appear to be spaced approximately 84 nm apart. The distance of 84 nm suggests that fibronectin is fully extended in fibrils and that the amino termini of adjacent fibronectin dimers overlap by 20 nm. As fibronectin fibrils become thicker, the average distance between EIIIA sites in fibronectin dimers decreases to 42 nm. This decrease in the distance between EIIIA sites may be due to a staggering of fibronectin dimers within the fibril as the fibril matures.

Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures.

Exogenous plasma and endogenous cellular fibronectins on the surface of cultured fibroblasts and in extracellular matrix fibrils were colocalized by fluorescent and high voltage immunoelectron microscopy. Fibroblast cultures grown in the presence or absence of cycloheximide were incubated with exogenous plasma fibronectin labeled with fluorescein isothiocyanate. A monoclonal antibody specific for the EIIIA sequence of cellular fibronectin was used to detect cellular fibronectin. A rabbit antifluorescein antibody identified fluoresceinated plasma fibronectin. In cultures incubated in the presence of cycloheximide, plasma fibronectin was bound to the cell surface and was assembled into extracellular fibrils. In cultures grown in the absence of cycloheximide, plasma and cellular fibronectins were observed in the same matrix fibrils and in the same locations on the cell surface. There was not, however, random admixture of the two proteins.