Children forgotten in hot cars: a mental models approach for improving public health messaging.

INTRODUCTION: On average, in the USA, 37 young children die every year due to vehicular heatstroke. Additionally, over half of these incidents occur when a parent/caregiver forgets a child in a vehicle. While various governmental and child safety advocacy groups have worked to raise awareness about these tragedies, rigorous studies have yet to be conducted that examine the current understanding and effectiveness of this public health messaging. METHODS: This study will employ a mental models approach in order to identify differences that exist between experts' and parents'/caregivers' knowledge and beliefs surrounding the topic of children forgotten in hot cars. We interviewed a diverse set of 25 parents/caregivers and seven experts in order to construct and explore these mental models. RESULTS: A comparative analysis was conducted, and three key differences were observed between these mental models. Unlike the experts, the parents/caregivers in the study emphasised perceived lifestyle factors (eg, low-income parent) as important elements in increasing an individual's likelihood of forgetting a child in a car. Importantly, the parents/caregivers primarily obtained information from news reports, while experts believed public health campaigns would reach more parents/caregivers. Lastly, while experts stressed that this tragedy could happen to anyone, most parents/caregivers failed to acknowledge that they could forget their own child in a car. CONCLUSIONS: To confront this denial, future public health messaging must strive to engage and reach all parents/caregivers. This can be accomplished using a multifaceted messaging strategy that includes personalising core messaging, providing additional resources to media outlets and building rapport between key partners.

Associations of HIV and prevalent type 2 diabetes mellitus in the context of obesity in South Africa.

It is unclear how rising obesity among people with HIV (PWH) in sub-Saharan Africa (SSA) impacts their risk of type 2 diabetes mellitus (diabetes). Using a South African national cross-sectional sample of adult PWH and their peers without HIV (PWOH), we examined the associations between HIV and prevalent diabetes across the spectrum of body mass index (BMI), waist circumference (WC) and waist-to-height ratio (WtHR). Analyses were sex stratified, and adjusted for age, sociodemographic and behavioral factors. The prevalence of diabetes among males was similar between PWH and PWOH, overall and at all levels of adiposity. In contrast, overall diabetes prevalence was higher among female PWOH than female PWH. However, there were differences according to adiposity such that, compared to female PWOH, relative diabetes prevalence in female PWH was reduced with obesity but accentuated with leanness. These differences in the relationship between adiposity and diabetes by HIV serostatus call for better mechanistic understanding of sex-specific adipose tissue biology in HIV in South Africa, and possibly in other HIV endemic settings in SSA.

Impaired glucose tolerance in mice with a targeted impairment of insulin action in muscle and adipose tissue.

Type 2 diabetes is a complex metabolic disorder characterized by peripheral insulin resistance and impaired beta cell function. Insulin resistance is inherited as a non-mendelian trait. In genetically predisposed individuals, resistance of skeletal muscle and adipose tissue to insulin action precedes the onset of clinical diabetes, and is thought to contribute to hyperglycaemia by leading to impaired beta cell function and increased hepatic glucose production. It is not clear whether beta cell and liver defects are also genetically determined. To test the hypothesis that insulin resistance in muscle and fat is sufficient to cause type 2 diabetes in the absence of intrinsic beta cell and liver abnormality, we generated transgenic mice that were insulin-resistant in skeletal muscle and adipose tissue. These mice developed all the prodromal features of type 2 diabetes but, despite the compounded effect of peripheral insulin resistance and a mild impairment of beta cell function, failed to become diabetic. These findings indicate the need for a critical re-examination of the primary site(s) of insulin resistance in diabetes.

Radiation hybrid map of the mouse genome.

Radiation hybrid (RH) maps are a useful tool for genome analysis, providing a direct method for localizing genes and anchoring physical maps and genomic sequence along chromosomes. The construction of a comprehensive RH map for the human genome has resulted in gene maps reflecting the location of more than 30,000 human genes. Here we report the first comprehensive RH map of the mouse genome. The map contains 2,486 loci screened against an RH panel of 93 cell lines. Most loci (93%) are simple sequence length polymorphisms (SSLPs) taken from the mouse genetic map, thereby providing direct integration between these two key maps. We performed RH mapping by a new and efficient approach in which we replaced traditional gel- or hybridization-based assays by a homogeneous 5'-nuclease assays involving a single common probe for all genetic markers. The map provides essentially complete connectivity and coverage across the genome, and good resolution for ordering loci, with 1 centiRay (cR) corresponding to an average of approximately 100 kb. The RH map, together with an accompanying World-Wide Web server, makes it possible for any investigator to rapidly localize sequences in the mouse genome. Together with the previously constructed genetic map and a YAC-based physical map reported in a companion paper, the fundamental maps required for mouse genomics are now available.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein.

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice.

In lipoatrophic diabetes, a lack of fat is associated with insulin resistance and hyperglycemia. This is in striking contrast to the usual association of diabetes with obesity. To understand the underlying mechanisms, we transplanted adipose tissue into A-ZIP/F-1 mice, which have a severe form of lipoatrophic diabetes. Transplantation of wild-type fat reversed the hyperglycemia, dramatically lowered insulin levels, and improved muscle insulin sensitivity, demonstrating that the diabetes in A-ZIP/F-1 mice is caused by the lack of adipose tissue. All aspects of the A-ZIP/F-1 phenotype including hyperphagia, hepatic steatosis, and somatomegaly were either partially or completely reversed. However, the improvement in triglyceride and FFA levels was modest. Donor fat taken from parametrial and subcutaneous sites was equally effective in reversing the phenotype. The beneficial effects of transplantation were dose dependent and required near-physiological amounts of transplanted fat. Transplantation of genetically modified fat into A-ZIP/F-1 mice is a new and powerful technique for studying adipose physiology and the metabolic and endocrine communication between adipose tissue and the rest of the body.

Enhanced glycosylation and sulfation of secretory proteoglycans is coupled to the expression of a basic secretory protein.

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

Natural history of retinopathy in children and young people with type 1 diabetes.

PurposeTo describe the prevalence and natural history of retinopathy in a cohort of children and young people with type 1 diabetes attending a tertiary hospital diabetes clinic.MethodsWe analysed retinopathy screening data from 2008 to 2010 on all eligible children using the 'Twinkle' diabetes database and the regional retinal screening database.ResultsA total of 88% (149/169) of eligible children were screened in 2008, median age 14 years, 52% male. The prevalence of retinopathy was 19.5% (30/149). All children had background retinopathy grade R1. There was significant difference in median (range) duration of diabetes, 7.7 years (0.6-13.7) vs 5 years (0.2-12.5) (P<0.001) and median (range) HbA1C, 9.1% (7.2-14) vs 8.6% (5.6-13.1) (P=0.02), between the groups with and without retinopathy. At 2- years follow-up, 12/30 (40%) had unchanged retinopathy grade R1, 10/30 (33.3%) showed resolution of changes (R0), 1/30 progressed to maculopathy, and 7/30 had no follow-up data. Median (range) HbA1C in 2008 and 2010 for the groups with stable vs resolved changes was similar, 9.1% (7.2-14.0) and 9.2% (7-14.0) vs 9.5% (7.8-14.0) and 9.2% (8.7-14.0). Of the 119 without retinopathy in 2008, 27 (22.5%) had developed retinopathy within 2 years, including 1 with pre-proliferative retinopathy and 1 with maculopathy. There was no significant difference in HbA1c between those who progressed to retinopathy (8.7% (7.1-13.1)) (8.7% (7.1-13.1)), and those who did not (8.6% (6.3-12.2)).ConclusionsPrevalence of background retinopathy in our cohort was comparable to the previously published reports, with higher HbA1c and longer duration of diabetes being significant risk factors. On short-term follow-up, Grade 1 retinopathy is likely to resolve in a third of patients and remain unchanged in just over a third.

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes.

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.

The purification and partial characterization of phospholipase A2, a secretory protein of rabbit parotid gland.

Phospholipase A2 (EC 3.1.1.4), a soluble enzyme present in secretion granule lysates and in the discharged secretion of rabbit parotid gland, has been purified by gel filtration. The enzyme preparations obtained from both lysates and secretion have been found to have identical amino acid compositions, amino terminal residues (Asx), isoelectric points (10.2) and electrophoretic behavior in polyacrylamide gels. The reduced and alkylated protein yields a single band upon electrophoresis in the presence of sodium dodecyl sulfate; the mobility corresponds to an apparent molecular weight of 16000. The enzyme is capable of action on phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol. Activity has been examined using as substrate sonicated vesicles consisting of PC and PE. Rates of hydrolysis have been determined by densitometry of lysophosphatides resolved and charred on thin-layer chromatograms. This approach has been used to follow enzyme purification, to indicate the preferential hydrolysis (approx. 2-fold) of PE vs. PC and to demonstrate that enzyme purified from granule lysates and discharged secretion shows the same heat stability and activity profile as a function of pH. A highly specific Ca2+ requirement for activity also has been identified for substances organized as phospholipid bilayers; the apparent inactivity of this enzyme within a Ca2+-containing storage organelle, the secretion granule, presents an interesting problem for future investigation.

Platelet phosphoprotein phosphatase activity. Its subcellular distribution and regulation.

Phosphoprotein phosphatase (EC 3.1.3.-) activity was found in human platelet homogenates and this activity was stimulated up to 20-fold by preincubation with trypsin. Both Mg2+ and Mn2+ greatly decreased the activity of trypsin-activated phosphatase but the activity of the untreated phosphatase was not affected by increasing the concentration of these divalent cations. It was also shown that the activity of the phosphatase underwent a transient inhibition upon addition of ATP and a permanent one with ATP-gamma-S.

Platelet phosphorylase kinase activity and its regulation by the calcium-dependent regulatory protein, calmodulin.

Platelet phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) was found to be a Ca2+-sensitive enzyme. It was two Ka values for Ca2+ viz. 0.25 and 2.6 microM, respectively. The "calcium-dependent regulator" or calmodulin can enhance the activity of phosphorylase kinase, increasing its affinity for Ca2+. In the presence of calmodulin phosphorylase kinase has only one, high affinity binding site for Ca2+ (Ka = 0.27 microM). Platelet phosphorylase kinase can be phosphorylated by endogenous cyclic AMP-dependent protein kinase increasing its catalytic activity and this activation process is reversed by dephosphorylation. The changing level of intracellular Ca2+ and cyclic AMP may control the activity of phosphorylase kinase, regulating the mobilization of glycogen.

Inheritance of restriction fragment length polymorphisms in Agaricus brunnescens.

The cultivated mushroom, Agaricus brunnescens, is secondarily homothallic; most basidia produce only two basidiospores, each of which receives two of the four post meiotic nuclei. The segregation of restriction fragment length polymorphisms (RFLPs) detected by four plasmid probes carrying single-copy nuclear DNA of Agaricus was followed in seven parental strains including commercial, wild-collected, and artificially synthesized heterokaryons. Of a total of 367 single-spore progeny examined, 351 (95.6%) were heteroallelic at all RFLP loci heteroallelic in the respective parents. Of the 16 segregant isolates, ten (2.7% of the total) were homoallelic at all segregating loci assayed, suggesting that these isolates were most probably derived from rare spores that had received only a single postmeiotic nucleus. Some of these ten isolates had recombinant genotypes. Only five isolates (1.4% of the total) showed homoallelism at one of the loci heteroallelic in the parent, while remaining heteroallelic at other loci. These five genotypes suggest that a crossover had occurred between a marker locus and its respective centromere. Taken together, the results suggest that meiosis in A. brunnescens is accompanied by low levels of recombination and that nonsister nuclei are preferentially incorporated into basidiospores after meiosis II.

Increased body fat mass and suppression of circulating leptin levels in response to hypersecretion of epinephrine in phenylethanolamine-N-methyltransferase (PNMT)-overexpressing mice.

Epinephrine is a major stress hormone that plays a central role in the control of metabolic function and energy homeostasis. To evaluate the role of epinephrine and the physiological and pathophysiological consequences of sustained elevation of epinephrine on metabolic and endocrine function, we studied several metabolic parameters and circulating leptin levels in a newly developed transgenic mouse model of phenylethanolamine-N-methyltransferase (PNMT) overexpression. A 100-fold overexpression of PNMT and subsequent elevation of epinephrine levels resulted in a marked suppression of circulating leptin levels in the transgenic animals (1.14 +/- 0.05 vs. 2.17 +/- 0.35 ng/ml; P < 0.01), which correlated negatively with plasma epinephrine (r = -0.82; P < 0.05), thus providing evidence for an inhibitory action of epinephrine on leptin production in vivo. In parallel, we found a marked increase in the body fat content of the transgenic animals (12.54 +/- 1.5 vs. 6.22 +/- 0.2%; P < 0.01) that was accompanied by enlarged adipocytes, indicating an increased lipid storage in PNMT transgenic mice. Interestingly, however, transgenic animals had normal body weight and did not exhibit major alterations in carbohydrate metabolism, as evidenced by analysis of random and fasted blood glucose levels, plasma insulin and C peptide levels, and insulin tolerance test. The metabolic alterations observed were not secondary to changes in food intake or increased activity of the hypothalamic-pituitary-adrenal axis, as there were no differences in these parameters. In summary, sustained primary overproduction of epinephrine resulted in suppression of plasma leptin levels and increased lipid storage in the PNMT transgenic mice. The concerted action of the sympathoadrenal system and reduced leptin may contribute to defending energy reservoirs while maintaining a normal body weight, which may be of vital importance under conditions of stress and energy deficiency.

Immunoglobulin-derived polypeptides enter the regulated secretory pathway in AtT-20 cells.

Constitutively secreted proteins have traditionally been believed to be excluded from the regulated secretory pathway. In this work we show that kappa light chain and Fc fragment, two markers of the constitutive pathway, are present in the regulated pathway in AtT-20 cells. They colocalize with the endogenous hormone ACTH and they exhibit stimulus-dependent secretion. The Fc fragment, which undergoes intracellular transport at the same rate as the ACTH precursor POMC, enters the forming secretory granules, however, it is partially lost during granule maturation. These observations show that classic constitutive secretory markers are not excluded from the regulated secretory pathway and that efficient sorting for regulated secretion occurs above a background of proteins which enter the granules without sorting.