Food models for portion size estimation of Asian foods.

BACKGROUND: Novel portion size estimation aids relevant to the cognitive potential of children are necessary for an improved accuracy in dietary recall. We developed graduated realistic food models for Asian foods and tested their accuracy and precision in children. METHODS: Food models were constructed for nine commonly consumed food items using a range of low cost materials. These were tested among a random sample of 80 school children (aged 10-16 years). A total of 719 estimations were made. RESULTS: A high percentage (68%) of correct estimations and high correlation (r > 0.95, P < 0.001) for estimated versus actual weight were observed. Bland-Altman analysis indicated 91% of estimations within the limits of agreement. Accuracy and precision was high for both amorphous and non-amorphous foods. CONCLUSIONS: Portion size estimation using realistic food models is found to be accurate and precise and is suitable for use in children.

Portion size estimation aids for Asian foods.

BACKGROUND: Portion size estimation is fundamental to the accuracy of dietary recall, as well as interventions in obesity. Data on portion size estimation aids (PSEA) for Asian foods are limited. PSEA for Asian foods were developed and their accuracy and precision were tested for inclusion in a food atlas. METHODS: Sixteen food items were selected to represent all food groups. Small and life size photographs were developed, and line diagrams were drawn. These, together with household utensils, were tested among a random sample of 80 schoolchildren (aged 10-16 years). A total of 3180 estimations were made: 876 for small photographs (n = 11 foods), 558 for life size photographs (n = 7 foods), 1271 for line diagrams (n = 16 foods) and 475 for household utensils (n = 6 foods). RESULTS: Line diagrams had a high percentage (63.9%) of correct estimations and a low percentage of over estimations (18.0%) and under estimations (18.1%), whereas household utensils performed poorly with 0.6% correct estimations. Greater accuracy and precision were obtained for amorphous foods with small photographs and for non-amorphous foods with line diagrams. The combination of small photographs (for vegetables) and line diagrams (for other foods) achieved a high correlation (r = 0.959, P ≤ 0.001), percentage correct estimations (68.3%) and low under estimations (19.9%) and over estimations (11.8%). Food texture, but not age or sex, was associated with correct estimations in all of the PSEA, except household utensils. CONCLUSIONS: Accuracy and precision of a combination PSEA is convincing, enabling inclusion into an Asian food atlas for dietary assessment and intervention.

Micronutrient status of female adolescent school dropouts.

OBJECTIVES: No data exists for nutritional status of female adolescent school dropouts despite one in seven adolescent girls in Sri Lanka being an early school leaver. The aim of this study was to assess the nutritional status of working and non-working female adolescent school dropouts. METHODS: A cross-sectional design was used to recruit 613 female adolescent school dropouts, aged 15-19 years, in two districts of the Western Province of Sri Lanka. BMI was calculated by assessment of weight and height. Haemoglobin, serum ferritin, serum folic acid, vitamin B12, and serum zinc were measured. RESULTS: When girls were grouped into age specific BMI categories, 32.8% of girls were underweight, while 6.1% were overweight. Prevalence of anaemia (haemoglobin <120 g/l) in the study population was 17 %. Low iron status (serum ferritin <20 µg/l) was noted in 29.4 % of girls, low serum folate in 28% (folic acid <3 µg/l) and zinc deficiency in 28.8% (zinc <66 µg/dl). Regression modeling indicated that dropping out of school early (at <14 years of age) was a significant risk factor for low serum ferritin (p=0.001, odds ratio=2.1). Working adolescents were at greater risk of low micronutrient status: low serum ferritin (p=0.009; odds ratio=1.8) serum folic acid (p=0.006; odds ratio=1.9) and zinc deficiency (p=0.001; odds ratio=2.1) than non-working adolescents. CONCLUSIONS: Dropping out of school early and being employed increases the risk of micronutrient deficiencies.

Association between early weight gain and later adiposity in Sri Lankan adolescents.

Early growth pattern is increasingly recognized as a determinant of later obesity. This study aimed to identify the association between weight gain in early life and anthropometry, adiposity, leptin, and fasting insulin levels in adolescence. A cross-sectional study was conducted in 366 school children aged 11-13 years. Weight, height, and waist circumference (WC) were measured. Fat mass (FM) was assessed using bioelectrical impedance analysis. Blood was drawn after a 12-h fast for insulin and leptin assay. Birth weight and weight at 6 months and at 18 months were extracted from Child Health Development Records. An increase in weight SD score (SDS) by ≥0.67 was defined as accelerated weight gain. Linear mixed-effects modeling was used to predict anthropometry, adiposity, and metabolic outcomes using sex, pubertal status, accelerated weight gain as fixed factors; age, birth weight, and family income as fixed covariates, and school as a random factor. Children with accelerated weight gain between birth and 18 months had significantly higher body mass index (BMI) SDS, WC SDS, height SDS, %FM, fat mass index (FMI), fat free mass index (FFMI), and serum leptin levels in adolescence. Accelerated weight gain between 6 and 18 months was associated with higher BMI SDS, WC SDS, %FM, and FMI, but not with height SDS or FFMI. Accelerated weight gain at 0-6 months, in children with low birth weight, was associated with higher height SDS, BMI SDS, WC SDS, %FM, and FMI; in children with normal birth weight, it was associated with BMI SDS, WC SDS, height SDS, and FFMI, but not with %FM or FMI. Effects of accelerated weight gain in early life on anthropometry and adiposity in adolescence varied in different growth windows. Accelerated weight gain during 6-18 months was associated with higher FM rather than linear growth. Effects of accelerated weight gain between 0 and 6 months varied with birth weight.

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility.

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.

Regulation of cell motility by mitogen-activated protein kinase.

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle.

The role of cyclic adenosine monophosphate-mediated phosphorylation of myosin light chain kinase in relaxing smooth muscle was examined. The kinase was immunoprecipitated from tissue extracts and the phosphate content was determined. The addition of forskolin to resting or methacholine-contracted muscles resulted in an increase in myosin light chain kinase phosphorylation of myosin light chain kinase is one of the reactions in the process by which cyclic adenosine monophosphate causes relaxation of smooth muscle.

Inhibition of myosin light chain kinase by p21-activated kinase.

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.

A myosin I isoform in the nucleus.

A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.

The Mechanobiology of the Actin Cytoskeleton in Stem Cells during Differentiation and Interaction with Biomaterials.

An understanding of the cytoskeleton's importance in stem cells is essential for their manipulation and further clinical application. The cytoskeleton is crucial in stem cell biology and depends on physical and chemicals signals to define its structure. Additionally, cell culture conditions will be important in the proper maintenance of stemness, lineage commitment, and differentiation. This review focuses on the following areas: the role of the actin cytoskeleton of stem cells during differentiation, the significance of cellular morphology, signaling pathways involved in cytoskeletal rearrangement in stem cells, and the mechanobiology and mechanotransduction processes implicated in the interactions of stem cells with different surfaces of biomaterials, such as nanotopography, which is a physical cue influencing the differentiation of stem cells. Also, cancer stem cells are included since it is necessary to understand the role of their mechanical properties to develop new strategies to treat cancer. In this context, to study the stem cells requires integrated disciplines, including molecular and cellular biology, chemistry, physics, and immunology, as well as mechanobiology. Finally, since one of the purposes of studying stem cells is for their application in regenerative medicine, the deepest understanding is necessary in order to establish safety protocols and effective cell-based therapies.

Activated PAK4 regulates cell adhesion and anchorage-independent growth.

The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.

Differential expression and functions of cortical myosin IIA and IIB isotypes during meiotic maturation, fertilization, and mitosis in mouse oocytes and embryos.

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

Evidence for isoforms of the phosphorylatable myosin light chain in rat uterus.

The phosphorylatable myosin light chain of rat uterus was resolved into four spots by two-dimensional gel electrophoresis. Antibody against the 20 kDa light chain from smooth muscle myosin recognized these four spots. Purified light chain exhibited four spots on a diagonal upon isoelectrofocusing in both first and second dimensions, proving that these spots are not due to artifactual charge modification. Accordingly, the four spots contain light chain isoforms derived from myosin.

Immunological properties of myosin light-chain kinases.

We have studied the immunological and structural properties of myosin light-chain kinases. Immunological studies were performed with affinity-purified antibodies to turkey gizzard smooth-muscle myosin light-chain kinase. Immunoprecipitation experiments demonstrated that avian smooth muscles contain a myosin light-chain kinase of Mr 130,000, whereas the enzyme immunoprecipitated from canine smooth muscles tested has an Mr of 150,000. These antibodies do not react with cardiac- or skeletal-muscle myosin light-chain kinases. Experiments performed with myosin light-chain kinases purified from turkey gizzards (Mr 130,000), bovine tracheal smooth muscle (Mr 160,000) and human platelets (Mr 100,000) demonstrated the following: the primary structures of the turkey gizzard and bovine tracheal enzymes appear to be quite different, based on one-dimensional peptide maps; only one-third as many antibodies bind to the bovine tracheal enzyme as compared to the turkey gizzard enzyme; the antibody:myosin light-chain kinase ratios for half-maximal inhibition of all three enzymes are similar. Based on these data, we conclude that myosin light-chain kinases constitute an immunologically and structurally heterogeneous group of enzymes that have certain catalytic and regulatory properties in common.

Protection from lung damage by blood filtration during deep hypothermia in puppies.

Ten puppies underwent deep hypothermia with surface cooling and bypass cooling. They were subjected to circulatory arrest for 30 minutes and then were rewarmed by means of bypass. There were 5 control puppies and 5 for which a Swank filter was placed in the arterial perfusion line. All puppies had some degree of lung damage, but it was more severe in those in which perfused blood was not filtered. The damage involved the lung vessels, interstitial tissue, and alveoli. Platelet-fibrin aggregates and margination and sequestration of leukocytes appeared to be important pathological findings. Apparently, the Swank blood filter protects the lung by removing leukocytes and damaged platelets from the circulation.

Immunoreactivity of paraffin-embedded normal tissues and mesenchymal tumors for smooth muscle myosin.

Immunohistochemical study of smooth muscle myosin, a protein distinct from skeletal, cardiac, or nonmyogenous myosins in paraffin-embedded normal tissues and benign and malignant mesenchymal tumors revealed its strong expression in normal smooth muscle, capillary endothelium, and pericytes. All benign smooth muscle tumors with exception of gastric leiomyomas and few other leiomyomas of the gastrointestinal tract displayed strong or moderate immunoreactivity. On the other hand, strong or moderate immunoreactivity was detected in only eight of 28 spindle-cell leiomyosarcomas, as well as in 13 out of 27 malignant fibrous histiocytomas and three out of nine malignant hemangiopericytomas, while epithelioid leiomyosarcomas, fibrosarcomas, malignant schwannomas, and synovial sarcomas were negative or only weakly positive. Our results demonstrate that, while smooth muscle myosin is a very good marker of normal smooth muscle and benign smooth muscle tumors, it is expressed in diagnostically significant amounts in less than a third of spindle-cell leiomyosarcomas and none of the studied epithelioid leiomyosarcomas.

cAMP, myosin dephosphorylation, and isometric relaxation of airway smooth muscle.

The temporal relationships among increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels, myosin dephosphorylation, and relaxation were investigated to clarify the mechanisms of airway muscle relaxation. Canine tracheal muscles isometrically contracted (82% of maximum force) with 10(-6) M methacholine were relaxed by adding either 4 x 10(-7) M atropine or 4 x 10(-5) M forskolin. Atropine had no effect on cAMP levels; myosin phosphorylation and force, however, decayed at the same rates and these two parameters returned to their basal pre-methacholine levels within 5 min. Forskolin treatment results in about a 10-fold increase in cAMP levels; myosin phosphorylation and force decayed simultaneously to their respective steady-state levels by 10 min but neither parameter returned to its pre-methacholine level. The addition of forskolin to muscles maximally contracted with 10(-4) M methacholine leads to about a 30-fold increase in cAMP levels. However, there are minimal decreases in myosin phosphorylation and force in these muscles. Thus myosin dephosphorylation appears to be essential for airway muscle relaxation, whereas an increase in cAMP in the absence of myosin dephosphorylation is insufficient to cause relaxation. Moreover, myosin dephosphorylation appears to be a common step in the cAMP-independent and cAMP-dependent mechanisms for airway muscle relaxation.

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle.

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.